EP1610816A2 - Mva virus expressing modified hiv envelope, gag, and pol genes - Google Patents

Mva virus expressing modified hiv envelope, gag, and pol genes

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Publication number
EP1610816A2
EP1610816A2 EP04758588A EP04758588A EP1610816A2 EP 1610816 A2 EP1610816 A2 EP 1610816A2 EP 04758588 A EP04758588 A EP 04758588A EP 04758588 A EP04758588 A EP 04758588A EP 1610816 A2 EP1610816 A2 EP 1610816A2
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Prior art keywords
gag
env
sequence
mva
appendix
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German (de)
French (fr)
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EP1610816B1 (en
Inventor
Bernard Moss
Patricia L. Earl
Linda Wyatt
Leigh Anne Eller
Thomas Vancott
Matthew Edward Harris
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GOVERNMENT OF THE UNITED STATES OF AMERICA, AS R
Government Of United States, A
Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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US Department of Health and Human Services
US Government
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Priority to EP10012380A priority Critical patent/EP2363143A3/en
Priority to EP10012381A priority patent/EP2371380A1/en
Priority to EP10012379A priority patent/EP2359851A3/en
Publication of EP1610816A2 publication Critical patent/EP1610816A2/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/53—DNA (RNA) vaccination
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
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    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • the invention provides modified vaccinia Ankara (MVA), a replication-deficient strain of vaccinia vims, expressing human immunodeficiency vims (HIV) env, gag, and pol genes.
  • VAA modified vaccinia Ankara
  • HAV human immunodeficiency vims
  • Ankara (rMVA) booster has controlled a highly pathogenic immunodeficiency vims challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency vims proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered seven months after the booster. These findings are envisioned as indicating that a relatively simple multiprotein DNA/MVA vaccine .can help to control the acquired immune deficiency syndrome (AIDS) epidemic. We also report that inoculations of rMVA induce good immune responses even without DNA priming. Brief Description ofthe Drawings
  • Figure 1 Phylo genetic relationships of HIV- 1 and HIV-2 based on identity of pol gene sequences. SrV cpz and SlVs mm are subhuman primate lentiviruses recovered from a chimpanzee and sooty mangabey monkey, respectively.
  • Figure 2. Phylogenetic relationships of HIV-1 groups M, N and O with four different SrV cpz isolates based on full-length pol gene sequences. The bar indicates a genetic distance of 0.1 (10% nucleotide divergence) and the asterisk positions group N HIV-1 isolates based on env sequences.
  • HTV-encoded proteins The location of the HTV genes, the sizes of primary translation products (in some cases polyproteins), and the processed mature viral proteins are indicated.
  • Figure 5 Schematic representation of a mature HIV-1 virion.
  • Figure 6. Linear representation of the HIV-1 Env glycoprotein. The arrow indicates the site of gpl60 cleavage to gpl20 and gp41. hi gpl20, cross-hatched areas represent variable domains (Vi to V 5 ) and open boxes depict conserved sequences (Ci to C 5 ). In the gp41 ectodomain, several domains are indicated: the N-terminal fusion peptide, and the two ectodomain helices (N- and C-helix). The membrane-spanning domain is represented by a black box.
  • FIG. 7 Temporal frequencies of Gag-specific T cells.
  • A Gag-specific CD8 T cell responses raised by DNA priming and rMVA booster immunizations.
  • the schematic presents mean Gag-CM9-tetramer data generated in the high-dose i.d. DNA-immunized animals.
  • B Gag-specific IFN- ⁇ ELISPOTs in A*01 (open bars) and non-A*01 (filled bars) macaques at various times before challenge and at two weeks after challenge. Three pools of 10 to 13 Gag peptides (22-mers overlapping by 12) were used for the analyses. The numbers above data bars represent the arithmetic mean ⁇ SD for the ELISPOTs within each group.
  • FIG. 8 Temporal viral loads, CD4 counts, and survival after challenge of vaccinated and control animals.
  • A Geometric mean viral loads and
  • B geometric mean CD4 counts.
  • C Survival curve for vaccinated and control animals. The dotted line represents all 24 vaccinated animals.
  • D Viral loads and
  • E CD4 counts for individual animals in the vaccine and control groups. The key to animal numbers is presented in (E). Assays for the first 12 weeks after challenge had a detection level of 1000 copies of RNA per milliliter of plasma. Animals with loads below 1000 were scored with a load of 500. For weeks 16 and 20, the detection level was 300 copies of RNA per milliliter. Animals with levels of virus below 300 were scored at 300.
  • A Geometric mean viral loads and
  • B geometric mean CD4 counts.
  • C Survival curve for vaccinated and control animals. The dotted line represents all 24 vaccinated animals.
  • D Viral loads and
  • E CD4 counts
  • Temporal tetramer + cells (dashed line) and viral loads (solid line).
  • B Ihtiacellular cytokine assays for IFN- ⁇ production in response to stimulation with the Gag-CM9 peptide at two weeks after challenge. This ex vivo assay allows evaluation of the functional status of the peak postchallenge tetramer "1" cells displayed in Figure 7A.
  • C Proliferation assay at 12 weeks after challenge. Gag-Pol-Env (open bars) and Gag-Pol (hatched bars) produced by transient tiansfections were used for stimulation. Supernatants from mock-transfected cultures served as control antigen.
  • Stimulation indices are the growth of cultures in the presence of viral antigens divided by the growth of cultures in the presence of mock antigen.
  • A Typical lymph node from a vaccinated macaque showing evidence of follicular hyperplasia characterized by the presence of numerous secondary follicles with expanded germinal centers and discrete dark and light zones.
  • B Typical lymph node from an infected contiol animal showing follicular depletion and paracortical lymphocellular atrophy.
  • C A representative lymph node from an age-matched, uninfected macaque displaying nonreactive germinal centers.
  • D The percentage ofthe total lymph node area occupied by germinal centers was measured to give a non-specific indicator of follicular hyperplasia. Data for uninfected controls are for four age-matched rhesus macaques.
  • FIG. 11 Temporal antibody responses. Micrograms of total Gag (A) or Env (B) antibody were determined with ELISAs. The titers of neutralizing antibody for SHTV-89.6 (C) and SHIV-89.6P (D) were determined with MT-2 cell killing and neutral red staining (D.C. Montefiori et al. 1988 J Gin Microbiol 26:231). Titers are the reciprocal of the serum dilution giving 50% neutralization of the indicated viruses grown in human PBMC. Symbols for animals are the same as in Figure 8. Figure 12.
  • the number at the upper right comer of each plot represents the frequency of tetramer-specific CD8 T cells as a % of total CD8 T cells.
  • the numbers above each column of F ACS data designate individual animals.
  • FIG. 13 Map of plasmid transfer vector pLW-48.
  • FIG. 14 A-I Sequences of plasmid transfer vector pLW-48, Psy II promoter (which controls ADA envelope expression), ADA envelope truncated, PmH5 promoter (which controls HXB2 gag pol expression), and HXB2 gag pol (with safety mutations, ⁇ integrase).
  • Figure 15 Plasmid transfer vector pLW-48 and making MVA recombinant virus MVA/HIV 48.
  • Figure 16. A clade B gag pol.
  • Figure 17 Sequence of new Psyn II promoter.
  • Figure 18. ⁇ LAS-1 and ⁇ LAS-2.
  • Figure 19. ⁇ LAS-1/UGDgag.
  • Figure 20. ⁇ LAS-2/XJGDenv.
  • Figure 21. pLAS-2/UGDrev env.
  • Figure 22 Schematic for recombinant MVA production.
  • Figure 23 Overview of making recombinant MVA/UGD viruses.
  • Figure 24 Immunoprecipitation analysis.
  • Figure 25 Functional analysis of expressed proteins.
  • A Virus-like partikle assay.
  • B Env fusion assay.
  • FIG. 26 MVA/UGD induced HIV env-and gag-specific antibody responses.
  • A HIV p24-specofoc serum IgG responses.
  • B HIV env-specific serum IgG responses.
  • C MVA UGD induced HIV env- and gag-specific antibody responses (study 1).
  • Figure 27 JVJVA/UGD induced gag-specific intracellular IFN- ⁇ production.
  • Figure 28A MVA/UGD induced gag-specific IFN- ⁇ ELISPOT.
  • Figure 28B MVA/UGD induced pol-specific IFN- ⁇ ELISPOT.
  • Figure 29 MVA/UGD induced gag-specific tetramer staining.
  • Figure 30 MVA/UGD induced gag-specific antibody responses (study 2).
  • Figure 31A & B MVA/UGD induced gag-and pol-specific intracellular IFN- ⁇ production (study 2).
  • Figure 33 MVA/UGD induced gag-specific tetramer staining (study 2).
  • Figure 34 JVTVA/UGD induced gag-specific cytotoxic T cell killing.
  • Figure 35 Immunoprecipitation analysis of cell lysates (MVA/KEA).
  • Figure 36 Gag particle assay (MVA/KEA).
  • Figure 37 Fusion assay (MVA/KEA).
  • Figure 38 MVA/KEA induced HIV-1 env-specific antibody responses.
  • FIG 39 MVA/KEA induced gag-specific intracellular IFN- ⁇ production.
  • Figure 40 MVA/KEA induced gag-specific tetramer staining.
  • Figure 41 MVA/KEA induced gag-specific IFN- ⁇ ELISPOT.
  • Figure 42 Immunoprecipitation analysis of cell lysates (MVA/TZC).
  • Figure 43 Fusion assay (MVA/TZC).
  • Vaccinia virus a member of the genus Orthopoxvirus in the family of Poxviridae, was used as live vaccine to immunize against the human smallpox disease.
  • Successful worldwide vaccination with vaccinia virus culminated in the eradication of variola virus, the causative agent of the smallpox ("The global eradication of smallpox. Final report of the global commission for the certification of smallpox eradication".
  • WHO declaration vaccination has been universally discontinued except for people at high risk of poxvirus infections (e.g. laboratory workers).
  • vaccinia viruses have also been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M. et al. 1982 PNAS USA 79:7415-7419; Smith, G.L. et al. 1984 Biotech Genet Engin Rev 2:383-407).
  • This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses.
  • the gene is integrated at a site in the viral DNA which is non- essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83,286 and No. 110,385).
  • the recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
  • modified vaccinia Ankara has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A. et al. 1975 Infection 3:6-14; Swiss Patent No. 568,392).
  • the MVA virus is publicly available from American Type Culture Collection as ATCC No. VR-1508.
  • MVA is distinguished by its great attenuation, that is to say by dimimshed virulence and ability to replicate in primate cells while maintaining good immunogenicity.
  • the MVA virus has been analyzed to dete ⁇ nine alterations in the genome relative to the parental CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). The resulting MVA virus became severely host cell restricted to avian cells.
  • MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr A. et al. 1978 Monshamteriol [B] 167:375-390; Stick! et al 1974 Dtsch Med Wschr 99:2386-2392). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine.
  • MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. 1992 PNAS USA 89:10847-10851). Additionally, novel vaccinia vector vaccines were established on the basis of MVA having foreign DNA sequences inserted at the site of deletion III within the MVA genome (Sutter, G. et al 1994 Vaccine 12:1032- 1040).
  • the recombinant MVA vaccinia viruses can be prepared as set out hereinafter.
  • a DNA-construct which contains a DNA-sequence which codes for a foreign polypeptide flanked by MVA DNA sequences adjacent to a naturally occurring deletion, e.g. deletion III, or other non-essential sites, within the MVA genome is introduced into cells infected with MVA, to allow homologous recombination.
  • Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker.
  • the DNA-construct to be inserted can be linear or circular.
  • a plasmid or polymerase chain reaction product is preferred.
  • the DNA-construct contains sequences flanking the left and the right side of a naturally occu ⁇ ing deletion, e.g. deletion III, within the MVA genome.
  • the foreign DNA sequence is inserted between the sequences flanking the naturally occu ⁇ ing deletion.
  • regulatory sequences which are required for the transcription of the gene, to be present on the DNA.
  • promoters are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385).
  • the DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al. 1973 Virol 52:456- 467; Wigler el al. 1979 Cell 16:777-785), by means of electroporation (Neumann et al. 1982 EMBO J 1:841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101:482-492), by means of liposomes (Stiaubinger et al. 1983 Meth Enzymol 101:512- 527), by means of spheroplasts (Schaffner 1980 PNAS USA 77:2163-2167) or by other methods known to those skilled in the art. HIVs and Their Replication
  • HIV-2 is more closely related to SIVs mm , a virus isolated from sooty mangabey monkeys in the wild, than to HIV-1. It is currently believed that HIV-2 represents a zoonotic tiansmission of SrV sram to man.
  • SIV CpZ A series of lentiviral isolates from captive chimpanzees, designated SIV CpZ , are close genetic relatives ofHrV-1. The earliest phylogenetic analyses of HIV-1 isolates focused on samples from
  • the M group of HTV-1 which includes over 95% ofthe global virus isolates, consists of at least eight discrete clades (A, B, C, D, F, G, H, and J), based on the sequence of complete viral genomes.
  • Members of HIV-l group O have been recovered from individuals living in Cameroon, Gabon, and Equatorial Guinea; their genomes share less than 50% identity in nucleotide sequence with group M viruses.
  • the more recently discovered group N HTV-1 strains have been identified in infected Cameroonians, fail to react sero logically in standard whole-virus enzyme-linked immunosorbent assay (ELISA), yet are readily detectable by conventional Western blot analysis.
  • ELISA enzyme-linked immunosorbent assay
  • HIV-1 genetic variation comes from studies of group M viruses of diverse geographic origin. Data collected during the past decade indicate that the HIV-1 population present within an infected individual can vary from 6% to 10% in nucleotide sequence. HIV-1 isolates within a clade may exhibit nucleotide distances of 15% in gag and up to 30% in gpl20 coding sequences. Interclade genetic variation may range between 30% and 40% depending on the gene analyzed. All of the H -l group M subtypes can be found in Africa. Clade A viruses are genetically the most divergent and were the most common HW-l subtype in Africa early in the epidemic.
  • clade C viruses have become the dominant subtype and now account for 48% of HW-l infections worldwide.
  • Clade B viruses the most intensively studied HW-l subtype, remain the most prevalent isolates in Europe and North America.
  • HW-l recombinants will be found in geographic areas such as Africa, South America, and Southeast Asia, where multiple HW-l subtypes coexist and may account for more than 10% of circulating HW-l strains. Molecularly, the genomes of these recombinant viruses resemble patchwork mosaics, with juxtaposed diverse HW-l subtype segments, reflecting the multiple crossover events contributing to their generation.
  • HW-l recombinants have arisen in Africa and a majority contains segments originally derived from clade A viruses, hi Thailand, for example, the composition of the predominant circulating strain consists of a clade A gag plus pol gene segment and a clade E env gene. Because the clade E env gene in Thai HW-l strains is closely related to the clade E env present in virus isolates from the Central African Republic, it is believed that the original recombination event occu ⁇ ed in Africa, with the subsequent introduction of a descendent virus into Thailand. Interestingly, no full-length HW-l subtype E isolate (i.e., with subtype E gag, pol, and env genes) has been reported to date.
  • HW-l isolates using the CXCR4 receptor are usually T cell line (TCL)-tiopic syncytium inducing (SI) strains, whereas those exclusively utilizing the CCR5 receptor (R5 viruses) are predominantly macrophage (M)-tiopic and non-syncytium inducing (NSI).
  • TCL T cell line
  • R5 viruses CCR5 receptor
  • M macrophage
  • NBI non-syncytium inducing
  • the dual-tropic R5/X4 strains which may comprise the majority of patient isolates and exhibit a continuum of tropic phenotypes, are frequently SI.
  • the three primary HW-l translation products are initially synthesized as polyprotein precursors, which are subsequently processed by viral or cellular proteases into mature particle-associated proteins (Fig. 4).
  • the 55-kd Gag precursor Pr55 Gag is cleaved into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins.
  • Retroviral Gag proteins are generally synthesized as polyprotein precursors; the HW-l Gag precursor has been named, based on its apparent molecular mass, Pr55 Gag .
  • the mRNA for Pr55 G g is the unspliced 9.2-kb transcript (Fig. 4) that requires Rev for its expression in the cytoplasm.
  • the viral protease (PR) cleaves Pr55 Gag during or shortly after budding from the cell to generate the mature Gag proteins pi 7 (MA), p24 (CA), p7 (NC), and p6 (see Fig. 4).
  • MA is localized immediately inside the lipid bilayer of the viral envelope
  • CA forms the outer portion of the cone-shaped core structure in the center of the particle
  • NC is present in the core in a ribonucleoprotein complex with the viral RNA genome (Fig. 5).
  • the HW Pr55 Gag precursor oligomerizes following its translation and is targeted to the plasma membrane, where particles of sufficient size and density to be visible by EM are assembled.
  • Formation of virus-like particles by Pr55 Gag is a self-assembly process, with critical Gag-Gag interactions taking place between multiple domains along the Gag precursor.
  • the assembly of virus-like particles does not require the participation of genomic RNA (although the presence of nucleic acid appears to be essential), jt? ⁇ -encoded enzymes, or Env glycoproteins, but the production of infectious virions requires the encapsidation ofthe viral RNA genome and the incorporation ofthe Env glycoproteins and the Gag-Pol polyprotein precursor Prl60 Gag"Po1 .
  • pol gene Downstream of gag lies the most highly conserved region of the HW genome, the pol gene, which encodes three enzymes: PR, RT, and IN (see Fig. 4).
  • RT and IN are required, respectively, for reverse transcription of the viral RNA genome to a double- stranded DNA copy, and for the integration of the viral DNA into the host cell chromosome.
  • PR plays a critical role late in the life cycle by mediating the production of mature, infectious virions.
  • the pol gene products are derived by enzymatic cleavage of a 160-kd Gag-Pol fusion protein, refe ⁇ ed to as Prl60 Ga ⁇ "Po1 . This fusion protein is produced by ribosomal frameshifting during translation of Pr55 Gag (see Fig.
  • the frame-shifting mechanism for Gag-Pol expression also utilized by many other retroviruses, ensures that the pol-detived proteins are expressed at a low level, approximately 5% to 10% that of Gag.
  • the N-terminus of Prl60 Gag"Po1 is myristylated and targeted to the plasma membrane.
  • retroviral Gag proteins are initially synthesized as polyprotein precursors that are cleaved to generate smaller products. Subsequent studies demonstrated that the processing function is provided by a viral rather than a cellular enzyme, and that proteolytic digestion of the Gag and Gag-Pol precursors is essential for virus infectivity. Sequence analysis of retroviral PRs indicated that they are related to cellular "aspartic" proteases such as pepsin and renin. Like these cellular enzymes, retroviral PRs use two apposed Asp residues at the active site to coordinate a water molecule that catalyzes the hydrolysis of a peptide bond in the target protein.
  • retroviral PRs function as true dimers.
  • X-ray crystallographic data from HW-l PR indicate that the two monomers are held together in part by a four-stranded antiparallel ⁇ -sheet derived from both N- and C- terminal ends of each monomer.
  • the substrate-binding site is located within a cleft formed between the two monomers.
  • the HW PR dimer contains flexible "flaps" that overhang the binding site and may stabilize the substrate within the cleft; the active-site Asp residues lie in the center of the dimer.
  • the primary sequences of retroviral PRs are highly divergent, yet their structures are remarkably similar.
  • Retroviral RTs have three enzymatic activities: (a) RNA-directed DNA polymerization (for minus-strand DNA synthesis), (b) RNaseH activity (for the degradation ofthe tRNA primer and genomic RNA present in DNA-RNA hybrid intermediates), and (c) DNA-directed DNA polymerization (for second- or plus-strand DNA synthesis).
  • the mature HW-l RT holoenzyme is a heterodimer of 66 and 51 kd subunits.
  • the 51-kd subunit (p51) is derived from the 66-kd (p66) subunit by proteolytic removal of the C-terminal 15-kd RNaseH domain of p66 by PR (see Fig. 4).
  • the crystal structure of HW- 1 RT reveals a highly asymmetric folding in which the orientations of the p66 and p51 subunits differ substantially.
  • the p66 subunit can be visualized as a right hand, with the polymerase active site within the palm, and a deep template-binding cleft formed by the palm, fingers, and thumb subdomains.
  • the polymerase domain is linked to RNaseH by the connection subdomain.
  • the active site located in the palm, contains three critical Asp residues (110, 185, and 186) in close proximity, and two coordinated Mg 2+ ions. Mutation of these Asp residues abolishes RT polymerizing activity.
  • the orientation of the three active-site Asp residues is similar to that observed in other DNA polymerases (e.g., the Klenow fragment of E. coli DNA poll).
  • the p51 subunit appears to be rigid and does not form a polymerizing cleft; Asp 110, 185, and 186 of this subunit are buried within the molecule.
  • the fingers adopt a more open configuration, thereby releasing the pyrophosphate and enabling RT to bind the next dNTP.
  • the structure of the HW-l RNaseH has also been determined by x-ray crystallography; this domain displays a global folding similar to that of E. coli RNaseH.
  • Integrase A distinguishing feature of retro virus replication is the insertion of a DNA copy of the viral genome into the host cell chromosome following reverse transcription.
  • the integrated viral DNA (the provirus) serves as the template for the synthesis of viral RNAs and is maintained as part of the host cell genome for the lifetime of the infected cell.
  • Retroviral mutants deficient in the ability to integrate generally fail to establish a productive infection.
  • Retroviral IN proteins are composed of three structurally and functionally distinct domains: an N-terminal, zinc-finger-containing domain, a core domain, and a relatively nonconserved C-terminal domain. Because of its low solubility, it has not yet been possible to crystallize the entire 288-amino-acid HW-l IN protein. However, the structure of all three domains has been solved independently by x-ray crystallography or NMR methods. The crystal structure of the core domain of the avian sarcoma virus IN has also been determined.
  • the N-terminal domain (residues 1 to 55), whose structure was solved by NMR spectioscopy, is composed of four helices with a zinc coordinated by amino acids His- 12, His- 16, Cys-40, and Cys-43.
  • the structure ofthe N-terminal domain is reminiscent of helical DNA binding proteins that contain a so-called helix-turn-helix motif; however, in the HW-l structure this motif contributes to dimer formation.
  • helix-turn-helix motif a so-called helix-turn-helix motif
  • this motif contributes to dimer formation.
  • poor solubility hampered efforts to solve the structure of the core domain.
  • attempts at crystallography were successful when it was observed that a Phe-to-Lys change at IN residue 185 greatly increased solubility without disrupting in vitro catalytic activity.
  • Each monomer of the HW-l IN core domain (IN residues 50 to 212) is composed of a five- stranded ⁇ -sheet flanked by helices; this structure bears striking resemblance to other polynucleotidyl transferases including RNaseH and the bacteriophage MuA transposase.
  • Three highly conserved residues are found in analogous positions in other polynucleotidyl transferases; in HW-l IN these are Asp-64, Asp-116 and Glu-152, the so-called D,D-35-E motif. Mutations at these positions block HW IN function both in vivo and in vitro.
  • the HW Env glycoproteins play a major role in the virus life cycle. They contain the determinants that interact with the CD4 receptor and coreceptor, and they catalyze the fusion reaction between the lipid bilayer of the viral envelope and the host cell plasma membrane. In addition, the HW Env glycoproteins contain epitopes that elicit immune responses that are important from both diagnostic and vaccine development perspectives.
  • the HW Env glycoprotein is synthesized from the singly spliced 4.3-kb Vpu/Env bicistronic mRNA (see Fig. 4); translation occurs on ribosomes associated with the rough endoplasmic reticulum (ER).
  • the 160-kd polyprotein precursor (gpl60) is an integral membrane protein that is anchored to cell membranes by a hydrophobic stop-transfer signal in the domain destined to be the mature TM Env glycoprotein, gp41 (Fig. 6).
  • the gpl60 is cotranslationally glycosylated, forms disulfide bonds, and undergoes oligomerization in the ER.
  • the predominant oligomeric form appears to be a trimer, although dimers and tetramers are also observed.
  • the gpl60 is transported to the Golgi, where, like other retroviral envelope precursor proteins, it is proteolytically cleaved by cellular enzymes to the mature SU glycoprotein gpl20 and TM glycoprotein gp41 (see Fig. 6).
  • the cellular enzyme responsible for cleavage of retroviral Env precursors following a highly conserved Lys/Arg-X-Lys/Arg-Arg motif is furin or a furin-like protease, although other enzymes may also catalyze gpl60 processing.
  • gpl 60 Cleavage of gpl 60 is required for Env-induced fusion activity and virus infectivity. Subsequent to gpl 60 cleavage, gpl 20 and gp41 form a noncovalent association that is critical for transport of the Env complex from the Golgi to the cell surface. The gpl20-gp41 interaction is fairly weak, and a substantial amount of gpl20 is shed from the surface of Env-expressing cells.
  • the HW Env glycoprotein complex in particular the SU (gpl 20) domain, is very heavily glycosylated; approximately half the molecular mass of gpl 60 is composed of oligosaccharide side chains.
  • oligosaccharide side chains During transport of Env from its site of synthesis in the ER to the plasma membrane, many of the side chains are modified by the addition of complex sugars.
  • the numerous oligosaccharide side chains form what could be imagined as a sugar cloud obscuring much of gpl20 from host immune recognition.
  • gpl20 contains interspersed conserved (Q to C 5 ) and variable (Vi to V 5 ) domains.
  • the Cys residues present in the gpl20s of different isolates are highly conserved and form disulfide bonds that link the first four variable regions in large loops.
  • a primary function of viral Env glycoproteins is to promote a membrane fusion reaction between the lipid bilayers of the viral envelope and host cell membranes. This membrane fusion event enables the viral core to gain entry into the host cell cytoplasm.
  • a number of regions in both g l20 and gp41 have been implicated, directly or indirectly, in Env-mediated membrane fusion.
  • C-terminal to the gp41 fusion peptide are two amphipathic helical domains (see Fig. 6) which play a central role in membrane fusion. Mutations in the N-terminal helix (refe ⁇ ed to as the N-helix), which contains a Leu zipper-like heptad repeat motif, impair infectivity and membrane fusion activity, and peptides derived from these sequences exhibit potent antiviral activity in culture.
  • the structure of the ectodomain of HW-l and SW gp41, the two helical motifs in particular, has been the focus of structural analyses in recent years.
  • gp41 adopts a hypothetical conformation that allows the fusion peptide to insert into the target lipid bilayer.
  • the formation of the gp41 six-helix bundle brings the viral and cellular membranes together and membrane fusion takes place.
  • the present invention relates to generation of a CD8 + T cell immune response against an antigen and also eliciting an antibody response. More particularly, the present invention relates to "prime and boost" immunization regimes in which the immune response induced by administration of a priming composition is boosted by administration of a boosting composition.
  • the present invention is based on inventors' experimental demonstration that effective boosting can be achieved using modified vaccinia Ankara (MVA) vectors, following priming with any of a variety of different types of priming compositions including recombinant MVA itself.
  • MVA modified vaccinia Ankara
  • T lymphocytes of the CD8 + type also known as cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • An important function of CD8 + cells is secretion of gamma interferon (IFN ⁇ ), and this provides a measure of CD8 T cell immune response.
  • IFN ⁇ gamma interferon
  • a second component ofthe immune response is antibody directed to the proteins ofthe pathogen.
  • the present invention employs MVA which, as the experiments described below show, has been found to be an effective means for providing a boost to a CD8 + T cell immune response primed to antigen using any of a variety of different priming compositions and also eliciting an antibody response.
  • the experimental work described below demonstrates that use of embodiments of the present invention allows for recombinant MVA virus expressing an HW antigen to boost a CD8 + T cell immune response primed by a DNA vaccine and also eliciting an antibody response.
  • the MVA was found to induce a CD8 + T cell response after intradermal, intramuscular or mucosal immunization.
  • Recombinant MVA has also been shown to prime an immune response that is boosted by one or more inoculations of recombinant MVA.
  • Non-human primates immunized with plasmid DNA and boosted with the MVA were effectively protected against intramucosal challenge with live virus.
  • a vaccination regime used intradermal, intramuscular or mucosal immunization for both prime and boost can be employed, constituting a general immunization regime suitable for inducing CD8 + T cells and also eliciting an antibody response, e.g. in humans.
  • the present invention in various aspects and embodiments employs an MVA vector encoding an HW antigen for boosting a CD8 + T cell immune response to the antigen primed by previous administration of nucleic acid encoding the antigen and also eliciting an antibody response.
  • a general aspect ofthe present invention provides for the use of an MVA vector for boosting a CD8 + T cell immune response to an HW antigen and also eliciting an antibody response.
  • One aspect of the present invention provides a method of boosting a CD8 + T cell immune response to an HW antigen in an individual, and also eliciting an antibody response, the method including provision in the individual of an MVA vector including nucleic acid encoding the antigen operably linked to regulatory sequences for production of antigen in the individual by expression from the nucleic acid, whereby a CD8 + T cell immune response to the antigen previously primed in the individual is boosted.
  • An immune response to an HW antigen may be primed by immunization with plasmid DNA or by infection with an infectious agent.
  • a further aspect of the invention provides a method of inducing a CD8 + T cell immune response to an HW antigen in an individual, and also eliciting an antibody response, the method comprising administering to the individual a priming composition comprising nucleic acid encoding the antigen and then administering a boosting composition which comprises an MVA vector including nucleic acid encoding the antigen operably linked to regulatory sequences for production of antigen in the individual by expression from the nucleic acid.
  • a further aspect provides for use of an MVA vector, as disclosed, in the manufacture of a medicament for administration to a mammal to boost a CD8 + T cell immune response to an HW antigen, and also eliciting an antibody response.
  • a medicament is 'generally for administration following prior administration of a priming composition comprising nucleic acid encoding the antigen.
  • the priming composition may comprise any viral vector, such as a vaccinia virus vector such as a replication-deficient strain such as modified vaccinia Ankara (MVA) or NYVAC (Tartaglia et al. 1992 Virology 118:217-232), an avipox vector such as fowlpox or canarypox, e.g. the strain known as ALVAC (Paoletti et al. 1994 Dev Biol Stand 82:65-69), or an adenovirus vector or a vesicular stomatitis virus vector or an alphavirus vector.
  • a viral vector such as a vaccinia virus vector such as a replication-deficient strain such as modified vaccinia Ankara (MVA) or NYVAC (Tartaglia et al. 1992 Virology 118:217-232), an avipox vector such as fowlpox or canarypox, e.g. the strain
  • the priming composition may comprise DNA encoding the antigen, such DNA preferably being in the form of a circular plasmid that is not capable of replicating in mammalian cells.
  • Any selectable marker should not be resistance to an antibiotic used clinically, so for example Kanamycin resistance is prefe ⁇ ed to Ampicillin resistance.
  • Antigen expression should be driven by a promoter which is active in mammalian cells, for instance the cytomegalovirus immediate early (CMV IE) promoter.
  • CMV IE cytomegalovirus immediate early
  • a priming composition is followed by boosting with a boosting composition, or first and second boosting compositions, the first and second boosting compositions being the same or different from one another.
  • Still further boosting compositions may be employed without departing from the present invention, hi one embodiment, a triple immunization regime employs DNA, then adenovirus as a first boosting composition, then MVA as a second boosting composition, optionally followed by a further (third) boosting composition or subsequent boosting administration of one or other or both of the same or different vectors.
  • DNA DNA then MVA then adenovirus, optionally followed by subsequent boosting administration of one or other or both ofthe same or different vectors.
  • the antigen may correspond to a complete antigen, or a fragment thereof.
  • Peptide epitopes or artificial strings of epitopes may be employed, more efficiently cutting out unnecessary protein sequence in the antigen and encoding sequence in the vector or vectors.
  • One or more additional epitopes may be included, for instance epitopes which are recognized by T helper cells, especially epitopes recognized in individuals of different HLA types.
  • An HW antigen of the invention to be encoded by a recombinant MVA virus includes polypeptides having immunogenic activity elicited by an amino acid sequence of an HW Env, Gag, Pol, Vif, Vpr, Tat, Rev, Vpu, or Nef amino acid sequence as at least one CD8 + T cell epitope.
  • This amino acid sequence substantially corresponds to at least one 10- 900 amino acid fragment and/or consensus sequence of a known HW Env or Pol; or at least one 10-450 amino acid fragment and/or consensus sequence of a l ⁇ iown HW Gag; or at least one 10-100 amino acid fragment and/or consensus sequence of a known HW Vif, Vpr, Tat, Rev, Vpu, or Nef.
  • Env is optionally deleted of subsequences. For example, regions of the gpl20 surface and gp41 transmembrane cleavage products can be deleted.
  • Gag is optionally deleted of subsequences.
  • regions of the matrix protein (pi 7), regions of the capsid protein (p24), regions of the nucleocapsid protein (pi), and regions of p6 (the C-terminal peptide ofthe Gag polyprotein) can be deleted.
  • a full length Pol precursor sequence is presented for use in the present invention, Pol is optionally deleted of subsequences.
  • regions of the protease protein (plO), regions of the reverse transcriptase protein (p66/p51), and regions of the integrase protein (p32) can be deleted.
  • Such an HW Env, Gag, or Pol can have overall identity of at least 50% to a known Env, Gag, or Pol protein amino acid sequence, such as 50-99% identity, or any range or value therein, while eliciting an immunogenic response against at least one strain of an HW.
  • Percent identify can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J Mol Biol 1970 48:443), as revised by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences.
  • the prefe ⁇ ed default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • an Env of the present invention is a variant form of at least one HW envelope protein.
  • the Env is composed of gpl 20 and the membrane-spanning and ectodomain of gp41 but lacks part or all of the cytoplasmic domain of gp41.
  • HW sequences are readily available from commercial and institutional HW sequence databases, such as GENBANK, or as published compilations, such as Myers et al eds., Human Retroviruses and AIDS, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Vol. I and II, Theoretical Biology and Biophysics, Los Alamos, N. Mex. (1993), or http://hiv-web.lanl.gov/.
  • Substitutions or insertions of an HW Env, Gag, or Pol to obtain an additional HW Env, Gag, or Pol, encoded by a nucleic acid for use in a recombinant MVA virus of the present invention can include substitutions or insertions of at least one amino acid residue (e.g., 1-25 amino acids).
  • at least one amino acid e.g., 1-25 amino acids
  • substitutions, insertions or deletions are identified based on safety features, expression levels, immunogenicity and compatibility with high replication rates of MVA.
  • Amino acid sequence variations in an HW Env, Gag, or Pol ofthe present invention can be prepared e.g., by mutations in the DNA.
  • Such HW Env, Gag, or Pol include, for example, deletions, insertions or substitutions of nucleotides coding for different amino acid residues within the amino acid sequence.
  • mutations that will be made in nucleic acid encoding an HW Env, Gag, or Pol must not place the sequence out of reading frame and preferably will not create complementary domains that could produce secondary mRNA structures.
  • HW Env, Gag, or Pol-encoding nucleic acid of the present invention can also be prepared by amplification or site-directed mutagenesis of nucleotides in DNA or RNA encoding an HW Env, Gag, or Pol and thereafter synthesizing or reverse transcribing the encoding DNA to produce DNA or RNA encoding an HW Env, Gag, or Pol, based on the teaching and guidance presented herein.
  • Recombinant MVA viruses expressing HW Env, Gag, or Pol of the present invention include a finite set of HW Env, Gag, or Pol-encoding sequences as substitution nucleotides that can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • HW Env, Gag, or Pol-encoding sequences as substitution nucleotides that can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • For a detailed description of protein chemistry and structure see Schulz, G.E. et al, 1978 Principles of Protein Structure, Springer-Verlag, New York, N.Y., and Creighton, T.E., 1983 Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, CA.
  • regulatory sequences for expression ofthe encoded antigen will include a natural, modified or synthetic poxvirus promoter.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA).
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • Terminator fragments include terminator fragments, polyadenylation sequences, marker genes and other sequences.
  • Other regulatory sequences including terminator fragments, polyadenylation sequences, marker genes and other sequences may be included as appropriate, in accordance with the knowledge and practice ofthe ordinary person skilled in the art: see, for example, Moss, B. (2001). Poxviridae: the viruses and their replication. In Fields Virology, D.M. Knipe, $ ⁇ d P.M. Howley, eds. (Philadelphia, Lippincott Williams & Wilkins), pp. 2849-2883.
  • Promoters for use in aspects and embodiments of the present invention must be compatible with poxvirus expression systems and include natural, modified and synthetic sequences.
  • Either or both of the priming and boosting compositions may include an adjuvant, such as granulocyte macrophage-colony stimulating factor (GM-CSF) or encoding nucleic acid therefor.
  • an adjuvant such as granulocyte macrophage-colony stimulating factor (GM-CSF) or encoding nucleic acid therefor.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • Administration of the boosting composition is generally about 1 to 6 months after administration ofthe priming composition, preferably about 1 to 3 months.
  • administration of priming composition, boosting composition, or both priming and boosting compositions is intradermal, intramuscular or mucosal immunization.
  • MVA vaccines may be achieved by using a needle to inject a suspension of the virus.
  • An alternative is the use of a needleless injection device to administer a virus suspension (using, e.g., BiojectorTM needleless injector) or a resuspended freeze-dried powder containing the vaccine, providing for manufacturing individually prepared doses that do not need cold storage. This would be a great advantage for a vaccine that is needed in rural areas of Africa.
  • MVA is a virus with an excellent safety record in human immunizations. The generation of recombinant viruses can be accomplished simply, and they can be manufactured reproducibly in large quantities. Intiadermal, intramuscular or mucosal administration of recombinant MVA virus is therefore highly suitable for prophylactic or therapeutic vaccination of humans against AIDS which can be controlled by a CD8 + T cell response.
  • the individual may have AIDS such that delivery ofthe antigen and generation of a CD8 + T cell immune response to the antigen is of benefit or has a therapeutically beneficial effect.
  • administration will have prophylactic aim to generate an immune response against HW or AIDS before infection or development of symptoms.
  • compositions may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. As noted, administration is preferably intradermal, intramuscular or mucosal.
  • Physiological saline solution dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Those of relevant skill in the art are well able to prepare suitable solutions using, for example, iso tonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as required.
  • a slow-release formulation may be employed.
  • the particles may be administered to an individual, particularly human or other primate.
  • Administration may be to another mammal, e.g. rodent such as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, dog or cat.
  • rodent such as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, dog or cat.
  • Administration is preferably in a "prophylactically effective amount" or a
  • therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, or in a veterinary context a veterinarian, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors l ⁇ iown to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, 1980, Osol, A. (ed.).
  • DNA is administered at a dose of 250 ⁇ g to 2.5 mg/injection, followed by MVA at a dose of 10 6 to 10 9 infectious virus particles/injection.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Delivery to a non-human mammal need not be for a therapeutic purpose, but may be for use in an experimental context, for instance in investigation of mechanisms of immune responses to an antigen of interest, e.g. protection against HW or AIDS.
  • SHW-89.6 The 89.6 chimera of simian and human immunodeficiency viruses (SHW-89.6) was used for the construction of immunogens and its highly pathogenic derivative, SHW-89.6P, for challenge (G.B. Karlsson et al. 1997 J
  • MVA Modified vaccinia Ankara
  • the DNA prime (DNA/89.6) expressed simian immunodeficiency virus (SW) Gag, Pol, Vif, Vpx, and Vpr and human immunodeficiency virus- 1 (HW-l) Env, Tat, and Rev from a single transcript (RJ. Gorelick et al. 1999 Virology 253:259; M.M. Sauter et al 1996 J Cell Biol 132:795).
  • SHW-89.6 sequences were cloned into the vector pGA2 using
  • pGA2 uses the CMV immediate early promoter without intron A and the bovine growth hormone polyadenylation sequence to express vaccine inserts.
  • Vaccine DNA was produced by Althea (San Diego, CA).
  • Althea San Diego, CA
  • DNA/89.6 produced about 300 ng of Gag and 85 ng of Env per 1x10 cells.
  • the rMVA booster (MVA/89.6) expressed SW Gag, Pol, and HW-l Env under the control of vaccinia virus early/late promoters.
  • the MVA double recombinant virus expressed both the HW 89.6 Env and the SW
  • Vaccination was accomplished by priming with DNA at 0 and 8 weeks and boosting with rMVA at 24 weeks (Fig. 7A).
  • I.d. and i.m. DNA immunizations were delivered in phosphate-buffered saline (PBS) with a needleless jet injector (Bioject, Portland, OR) to deliver five i.d. 100- ⁇ l injections to each outer thigh for the 2.5-mg dose of DNA or one i.d. 100- ⁇ l injection to the right outer thigh for the 250- ⁇ g dose of plasmid.
  • I.m. deliveries of DNA were done with one 0.5-ml injection of DNA in PBS to each outer thigh for the 2.5-mg dose and one 100- ⁇ l injection to the right outer thigh for the 250- ⁇ g dose.
  • lxlO 8 pfu of MVA/89.6 was administered both i.d. and i.m. with a needle.
  • One 100- ⁇ l dose was delivered to each outer thigh for the i.d. dose and one 500- ⁇ l dose to each outer thigh for the i.m dose.
  • Control animals received 2.5 mg ofthe pGA2 vector without vaccine insert with the Bioject device to deliver five 100- ⁇ l doses i.d. to each outer thigh.
  • the control MVA booster immunization consisted of 2xl0 8 pfu of MVA without an insert delivered i.d. and i.m. as described for MVA/89.6.
  • Animal numbers are as follows: 1, RBr-5*; 2, Rfrn-5*; 3, RQf-5*; 4, RZe-5; 5, ROm-5; 6, RDm-5; 7, RAj-5*; 8, RJi-5*; 9, RA1-5*; 10, RDe-5*; 11, RAi-5; 12, RPr-5; 13, RKw-4*; 14, RWz-5*; 15, RGo-5; 16, RLp-4; 17, RWd-6; 18, RAt-5; 19, RPb-5*; 20, RIi-5*; 21, RIq-5; 22, RSp-4; 23, RSn-5; 24, RGd-6; 25, RMb-5*; 26, RGy-5*; 27, RUs-4; and 28, RPm-5. Animals with the A*01 ⁇ allele are indicated with asterisks.
  • D ⁇ A priming followed by rMVA boosting generated high frequencies of virus- specific T cells that peaked at one week following the rMVA booster (Fig. 7).
  • the frequencies of T cells recognizing the Gag-CM9 epitope were assessed by means of Mamu- A*01 tetramers, and the frequencies of T cells recognizing epitopes throughout Gag were assessed with pools of overlapping peptides and an enzyme-linked immunospot (ELISPOT) assay (CA. Power et al. 1999 J Immunol Methods 227:99).
  • ELISPOT enzyme-linked immunospot
  • PBMC peripheral blood mononuclear cells
  • FITC fluorescein isothiocyanate
  • PerCP CD8 conjugated to peridinin chlorophyl protein
  • SEQ ID NO: 6 Gag-CM9 conjugated to allophycocyanin (APC), in a volume of 100 ⁇ l at 8° to 10°C for 30 min.
  • Cells were washed twice with cold PBS containing 2% fetal bovine serum (FBS), fixed with 1% paraformaldehyde in PBS, and analyzed within 24 hrs on a FACScaliber (Becton Dickinson, San Jose, CA). Cells were initially gated on lymphocyte populations with forward scatter and side scatter and then on CD3 cells. The CD3 cells were then analyzed for CD8 and tetramer-binding cells. About 150,000 lymphocytes were acquired for each sample. Data were analyzed using Flo Jo software (Tree Star, San Carlos, CA).
  • IFN- ⁇ ELISPOTs For interferon- ⁇ (IFN- ⁇ ) ELISPOTs, MULTISCREEN 96 well filtration plates (Millipore Inc. Bedford, MA) were coated overnight with antibody to human IFN- ⁇ (Clone B27, Pharmingen, San Diego, CA) at a concentration of 2 ⁇ g/ml in sodium bicarbonate buffer (pH 9.6) at 8° to 10°C. Plates were washed two times with RPMI medium and then blocked for 1 hour with complete medium (RPMI containing 10% FBS) at 37°C. Plates were washed five more times with plain RPMI medium, and cells were seeded in duplicate in 100 ⁇ l complete medium at numbers ranging from 2xl0 4 to 5xl0 5 cells per well.
  • IFN- ⁇ interferon- ⁇
  • Peptide pools were added to each well to a final concentration of 2 ⁇ g/ml of each peptide in a volume of 100 ⁇ l in complete medium.
  • Cells were cultured at 37°C for about 36 hrs under 5% CO 2 . Plates were washed six times with wash buffer (PBS with 0.05% Tween-20) and then incubated with 1 ⁇ g of biotinylated antibody to human IFN- ⁇ per milliliter (clone 7- 86-1; Diapharma Group, West Chester, OH) diluted in wash buffer containing 2% FBS. Plates were incubated for 2 hrs at 37°C and washed six times with wash buffer.
  • wash buffer PBS with 0.05% Tween-20
  • ELISPOTs The frequencies of ELISPOTs are approximate because different dilutions of cells have different efficiencies of spot formation in the absence of feeder cells (CA. Power et al. 1999 J Immunol Methods 227: 99). The same dilution of cells was used for all animals at a given time point, but different dilutions were used to detect memory and acute responses.
  • Gag-CM9 tetramer analyses were restricted to macaques that expressed the Mamu- A*01 histocompatibility type, whereas ELISPOT responses did not depend on a specific histocompatibility type.
  • the DNA immunizations raised low levels of memory cells that expanded to high frequencies within 1 week ofthe rMVA booster (Fig. 7 and 12).
  • CD8 cells specific to the Gag-CM9 epitope expanded to frequencies as high as 19% of total CD8 T cells (Fig. 12). This peak of specific cells underwent a 10- to 100-fold contraction into the DNA/MVA memory pool (Fig. 7A and 12).
  • ELISPOTs for three pools of Gag peptides also underwent a major expansion (frequencies up to 4000 spots for lxlO 6 PBMC) before contracting from 5- to 20-fold into the DNA/MVA memory response (Fig. 7B).
  • the frequencies of ELISPOTs were the same in macaques with and without the A *01 histocompatibility type (P>0.2).
  • Simple linear regression was used to estimate co ⁇ elations between postbooster and postchallenge ELISPOT responses, between memory and postchallenge ELISPOT responses, and between logarithmically tiansformed viral loads and ELISPOT frequencies. Comparisons between vaccine and control groups and ⁇ 4*0i and non A*01 macaques were performed by means of two-sample t tests with logarithmically transformed viral load and ELISPOT responses. Two-way analyses of variance were used to examine the effects of dose and route of administration on peak DNA/MVA ELISPOTs, on memory DNA/MVA ELISPOTs, and on logarithmically transformed Gag antibody data.
  • the rank order for the height of the ELISPOTs in the vaccine groups was 2.5 mg i.d. > 2.5 mg i.m. > 250 ⁇ g i.d. > 250 ⁇ g i.m. (Fig. 7B).
  • the IFN- ⁇ ELISPOTs included both CD4 and CD8 cells. Gag- CM9-specific CD8 cells had good lytic activity after restimulation with peptide.
  • the highly pathogenic SHW-89.6P challenge was administered intrarectally at 7 months after the rMVA booster, when vaccine-raised T cells were in memory (Fig. 7).
  • the challenge stock (5.7 x 10 9 copies of viral RNA per milliliter) was produced by one intravenous followed by one intrarectal passage in rhesus macaques of the original SHW-89.6P stock (G.B. Karlsson et al. 1997 J Virol 71:4218). Lymphoid cells were harvested from the intrarectally infected animal at peak viremia, CD8-depleted, and mitogen-stimulated for stock production.
  • a feeding tube (8Fr (2.7 mm) x 16 inches (41 cm); Sherwood Medical, St. Louis, MO) was inserted into the rectum for a distance of 15 to 20 cm.
  • a syringe containing 20 intrarectal infectious doses in 2 ml of RPMI- 1640 plus 10% FBS was attached to the tube and the inoculum was slowly injected into the rectum.
  • the feeding tube was flushed with 3.0 ml of RPMI without FBS and then slowly withdrawn.
  • RNA from 150 ⁇ l of ACD anticoagulated plasma was directly extracted with the QIAamp Viral RNA kit (Qiagen), eluted in 60 ⁇ l of AVE buffer, and frozen at -80°C until SHW RNA quantitation was performed.
  • dNTP deoxynucleoti.de triphosphate
  • the reaction mix was adjusted to a final volume of 50 ⁇ l containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 4 mM MgCl 2 , 0.4 mM each dNTP, 0.2 ⁇ M forward primer, 0.2 ⁇ M reverse primer, 0.1 ⁇ M probe, and 5 units AmpliTaq Gold DNA polymerase (all reagents from PerkinElmer Applied Biosystems, Foster City, CA).
  • the primer sequences witliin a conserved portion of the SW gag gene are the same as those described previously (S. Staprans et al. in: Viral Genome Methods K. Adolph, ed. CRC Press, Boca Raton, FL, 1996 pp.
  • PCR product accumulation was monitored with the 7700 sequence detector and a probe to an internal conserved gag gene sequence: 6FAM- CTGTCTGCGTCATTTGGTGC-Tamra (SEQ ID NO: 8), where FAM and Tamra denote the reporter and quencher dyes.
  • SHW RNA copy number was determined by comparison with an external standard curve consisting of virion-derived SWmac239 RNA quantified by the SW bDNA method (Bayer Diagnostics, Emeryville, CA).
  • the intraassay coefficient of variation was ⁇ 20% for samples containing >10 4 SHW RNA copies per milliliter, and ⁇ 25% for samples containing 10 3 to 10 4 SHW RNA copies per milliliter.
  • both high-dose DNA-primed groups and the low-dose i.d. DNA-primed group had reduced their geometric mean loads to about 1000 copies of viral RNA per milliliter.
  • the low-dose i.m. DNA-primed group had a geometiic mean of 6x10 copies of viral RNA and the nonvaccinated contiols had a geometric mean of 2 x 10 6 .
  • the low-dose i.m. group had reduced its geometric mean copies of viral RNA to 1000.
  • the 24 vaccinated animals only one animal, animal number 22 in the low-dose i.m. group, had intermittent viral loads above IxlO 4 copies per milliliter (Fig 8D).
  • Fig. 7 and 9A At one week after challenge, the frequency of tetramer cells in the peripheral blood had decreased, potentially reflecting the recruitment of specific T cells to the site of infection (Fig. 9A). However, by two weeks after challenge, tetramer "1" cells in the peripheral blood had expanded to frequencies as high as, or higher than, after the rMVA booster (Fig. 7 and 9A). The majority of the tetramer "1" cells produced IFN- ⁇ in response to a 6-hour peptide stimulation (Fig. 9B) (S.L. Waldrop et al. 1997 J Gin Invest 99:1739) and did not have the "stunned" IFN- ⁇ negative phenotype sometimes observed in viral infections (F. Lechner et al.
  • CTPYDTNQM Gag-CM9 peptide
  • SEQ ID NO: 6 Gag-CM9 peptide
  • RPMI RPMI containing 10% FBS and monensin (10 ⁇ g/ml) was added, and the cells were cultured for an additional 5 hrs at 37°C at an angle of 5° under 5% CO 2 .
  • Cells were surface stained with antibodies to CD8 conjugated to PerCP (clone SKI, Becton Dickinson) at 8° to 10°C for 30 min, washed twice with cold PBS containing 2% FBS, and fixed and permeabilized with Cytofix/Cytoperm solution (Pharmingen).
  • the tetramer 4" cells in the control group like those in the vaccine group, produced IFN- ⁇ after peptide stimulation (Fig. 9B).
  • Fig. 9B The tetramer 4" cells in the control group, like those in the vaccine group, produced IFN- ⁇ after peptide stimulation (Fig. 9B).
  • three of the four controls had undetectable levels of IFN- ⁇ -producing ELISPOTs. This rapid loss of antiviral T cells in the presence of high viral loads may reflect the lack of CD4 help.
  • T cell proliferative responses demonstrated that virus-specific CD4 cells had survived the challenge and were available to support the antiviral immune response (Fig. 9C).
  • About 0.2 million PBMC were stimulated in triplicate for 5 days with the indicated antigen in 200 ⁇ l of RPMI at 37°C under 5% CO 2 .
  • Supernatants from 293T cells transfected with DNA expressing either SHW-89.6 Gag and Pol or SHW-89.6 Gag, Pol and Env were used directly as antigens (final concentration of ⁇ 0.5 ⁇ g of p27 Gag per milliliter).
  • Supernatants from mock DNA (vector alone) ⁇ transfected cells served as negative controls.
  • Stimulation indices are the counts of tritiated thymidine incorporated in PBMC stimulated with 89.6 antigens divided by the counts of tritiated thymidine incorporated by the same PBMC stimulated with mock antigen.
  • mean stimulation indices for Gag-Pol-Env or Gag-Pol proteins ranged from 35 to 14 in the vaccine groups but were undetectable in the control group. Consistent with the proliferation assays, intracellular cytokine assays demonstrated the presence of virus-specific CD4 cells in vaccinated but not control animals.
  • the overall rank order ofthe vaccine groups for the magnitude ofthe proliferative response was 2.5 mg i.d. > 2.5 mg i.m. > 250 ⁇ g i.d. > 250 ⁇ g i.m.
  • lymph nodes from the vaccinated animals were morphologically intact and responding to the infection, whereas those from the infected controls had been functionally destroyed (Fig. 10).
  • Nodes from vaccinated animals contained large numbers of reactive secondary follicles with expanded germinal centers and discrete dark and light zones (Fig. 10A).
  • lymph nodes from the nonvaccinated control animals showed follicular and paracortical depletion (Fig. 10B), while those from unvaccinated and unchallenged animals displayed normal numbers of minimally reactive ge ⁇ ninal centers (Fig. 10C).
  • Germinal centers occupied ⁇ 0.05% of total lymph node area in the infected controls, 2% of the lymph node area in the uninfected controls, and up to 18% ofthe lymph node area in the vaccinated groups (Fig. 10D). More vigorous immune reactivity in the low-dose than the high-dose DNA-primed animals was suggested by more extensive germinal centers in the low dose group (Fig. 10D).
  • in situ hybridization for viral RNA revealed rare virus-expressing cells in lymph nodes from 3 of the 24 vaccinated macaques, whereas virus-expressing cells were readily detected in lymph nodes from each o the infected control animals.
  • the cytomorphology of infected lymph node cells was consistent with a macrophage phenotype.
  • the prime/boost strategy raised low levels of antibody to Gag and undetectable levels of antibody to Env (Fig. 11).
  • Postchallenge antibodies to both Env and Gag underwent anamnestic responses with total Gag antibody reaching heights approaching 1 mgJm and total Env antibody reaching heights of up to 100 ⁇ g/ml.
  • Enzyme-linked immimosorbent assays for total antibody to Gag used bacterially produced SIV gag p27 to coat wells (2 ⁇ g per milliliter in bicarbonate buffer).
  • the DNA/MVA vaccine controlled the infection, rapidly reducing viral loads to near or below 1000 copies of viral RNA per milliliter of blood. Containment, rather than prevention of infection, affords the opportunity to establish a chronic infection (H.L. Robinson et al. 1999 Nat Med 5:526). By rapidly reducing viral loads, a multiprotein DNA/MVA vaccine will extend the prospect for long-term non-progression and limit HW transmission (J.W. Mellors et al. 1996 Science 272:1167; T.C. Quinn et al. 2000 NEngl J Med 342:921). EXAMPLE 2
  • MVA Expressing Modified HIV Env, Gag, and Pol Genes This disclosure describes the construction of a modified vaccinia Ankara (MVA) recombinant virus, MV A/HIV clade B recombinant virus expressing the HW strain ADA env and the HXB2 gag pol (MVA/HW ADA env + HXB2 gag pol).
  • MVA modified vaccinia Ankara
  • MVAJHTV 48 the lab name of MVAJHTV 48 will be used, which denotes the plasmid from which the construct comes.
  • the HW gag-pol genes were derived from the Clade B infectious HXB2 virus.
  • the gag-pol gene was truncated so that most of the integrase coding sequences were removed and amino acids 185, 266, and 478 were mutated to inactivate reverse transcriptase, inhibit strand transfer activity, and inliibit the RNaseH activity, respectively.
  • the Clade B CCR5 tropic envelope gene was derived from the primary ADA isolate; TTTTTNT (SEQ ID NO: 14) sequences were mutated without changing coding capacity to prevent premature transcription termination and the cytoplasmic tail was truncated in order to improve surface expression, immunogenicity, and stability of the MVA vector.
  • the HW genes were inserted into a plasmid transfer vector so that gag-pol gene was regulated by the modified H5 early/late vaccinia virus promoter and the env gene was regulated by the newly designed early/late Psyn II promoter to provide similar high levels of expression.
  • a self- deleting GUS reporter gene was included to allow detection and isolation of the recombinant virus.
  • the HW genes were flanked by MVA sequences to allow homologous recombination into the deletion 3 site so that the recombinant MVA would remain TK positive for stability and high expression in resting cells.
  • the recombinant MVA was isolated and shown to express abundant amounts of gag-pol-env and to process gag. Production of HW-like particles was demonstrated by centrifugation and by electron microscopy. The presence of env in the HW-like particles was demonstrated by immunoelectron microscopy.
  • the plasmid transfer vector used to make the MVA recombinant virus, pLW-48, ( Figure 15) by homologous recombination was constructed as follows:
  • flanking areas on either side of deletion III designated flank 1 and flank 2, containing 926 and 520 base pairs respectively, were amplified by PCR from the MVA stains of vaccinia virus.
  • a promoter, the mH5 which had been modified from the originally published sequence by changing two bases that had been shown by previously published work to increase the expression ofthe cloned gene, was added.
  • a clade B gag pol ( Figure 16) was truncated so that the integrase was removed and was cloned into the plasmid so that it was controlled by the mH5 promoter.
  • This gene contained the complete HXB2 sequence o the gag.
  • the pol gene has reverse transcriptase safety mutations in amino acid 185 within the active site of RT, in amino acid 266 which inhibits strand transfer activity, and at amino acid 478 which inhibits the RNaseH activity, hi addition, the integrase gene was deleted past EcoRI site.
  • the pll promoter and GUS reporter gene were added between the two direct repeats of flank 1 so that this screening marker could initially be used for obtaining the recombinant virus, yet deleted out in the final recombinant virus (Scheiflinger, F. et al. 1998 Arch Virol 143:467-474; Carroll, M.W. and B. Moss 1995 BioTechniques 19:352- 355).
  • a truncated version of the ADA envelope with a silent 5TNT mutation was obtained by PCR and inserted in the plasmid under the control of the Psyn II promoter.
  • the envelope was truncated in the cytoplasmic tail of the gp41 gene, deleting 115 amino acids of the cytoplasmic tail. This truncation was shown to increase the amount of envelope protein on the surface of infected cells and enhance immunogenicity of the envelope protein in mice, and stability ofthe recombinant virus in tissue culture.
  • MVA virus which may be obtained from ATCC Number VR-1508, was plaque purified three times by terminal dilutions in chicken embryo fibroblasts (CEF), which were made from 9 day old SPF Premium SPAFAS fertile chicken eggs, distributed by B and E Eggs, Stevens, PA.
  • CEF chicken embryo fibroblasts
  • Secondary CEF cells were infected at an MOI of 0.05 of MVA and transfected with 2 ⁇ g of pLW-48, the plasmid described above. Following a two-day incubation at
  • the virus was harvested, frozen and thawed 3x, and plated out on CEF plates.
  • the recombinant virus was shown to produce gag particles in the supernatant of infected cells by pelleting the 3s S-labeled particles on a 20% sucrose cushion.
  • Gag particles were also visualized both outside and budding from cells as well as within vacuoles of cells in the electron microscope in thin sections. These gag particles had envelope protein on their surface.
  • nucleotide sequences determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to
  • nucleotide sequence at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
  • the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well l ⁇ iown in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that
  • the MVA recombinant virus is made using a transiently expressed GUS marker that is deleted in the final virus.
  • High expression of the ADA envelope is possible because of a new hybrid early/late promoter, Psyn II.
  • the envelope has been truncated because we have shown truncation of the envelope enhances the amount of protein on the surface of the infected cells, and hence enhances immunogenicity; stability of the recombinant is also enhanced.
  • the MVA recombinant makes gag particles which have been shown by pelleting the particles through sucrose and analyzing by PAGE. Gag particles with envelope protein on the surface have also been visualized in the electron microscope.
  • Additional modified or synthetic promoters were designed for gene expression in MVA or other poxviruses. Promoters were modified to allow expression at early and late times after infection and to reduce possibility of homologous recombination between identical sequences when multiple promoters are used in same MVA vector. Promoters are placed upstream of protein coding sequence.
  • m7.5 promoter SEQ ID NO: 10: CGCTTTTT ATAGTAAGTTTTTC ACCC ATAAATAATAAAT ACAAT AATT AATTTCT CGTAAAAATTGAAAAACTATTCTAATTTATTGCACGGT
  • Psyn II promoter (SEQ ID NO: 2): TAAAAAATGAAAAAATATTCTAATTTATAGGACGGTTTTGATTTTCTTTTTTTCT
  • Table A MVA/48 immunization - guinea pigs.
  • Groups of guinea pigs were immunized at days 0 and 30 with 1 xlO 8 infectious units of MVA/48 by either the intramuscular (EVf) or intradermal (ID) route.
  • EVf intramuscular
  • ID intradermal
  • Sera taken before as well as after each immunization were analyzed for neutralizing activity against HW-l-MN. Titers are the reciprocal serum dilution at which 50% of MT-2 cells were protected from virus-induced killing. Significant neutralizing activity was observed in all animals after the second immunization with MVA/48 (day 49).
  • Table B Frequencies of HW-l gag-specific T cells following immunization of mice with MVA/48.
  • mice were immunized at days 0 and 21 with 1 xlO 7 infectious units of MVA 48 by one of three routes: intraperitoneal (IP), intradermal (ID), or intramuscular (TM).
  • IP intraperitoneal
  • ID intradermal
  • TM intramuscular
  • a control group was immunized with non-recombinant MVA.
  • splenocytes were prepared and stimulated in vitro with an immunodominant peptide from HW-l p24 for 7 days. The cells were then mixed either with peptide-pulsed P815 cells or with soluble peptide.
  • Gamma interferon-producing cells were enumerated in an ELISPOT assay. A value of >500 was assigned to wells containing too many spots to count. Strong T cell responses have been reported in mice immunized IP with other viruses. In this experiment, IP immunization of mice with MVA/48 elicited very strong HW-l gag-specific T cell responses.
  • Table C DNA prime and MVA/48 boost - total ELISPOTS per animal.
  • PBMC peripheral blood mononuclear cells
  • ELISPOT assay as follows: PBMC were incubated for 30-36 hours in the presence of pools of overlapping peptides corresponding to the individual HW-l antigens in the vaccines. The total number of gamma interferon-producing cells from each animal is shown in the table. T cell responses to DNA vaccination were limited (weeks 2-20). However, boosting with MVA 48 resulted in very strong HW-l -specific T cell responses in all animals (week 25).
  • Table D Antibody response following immunization of macaques with MVA/SHW KB9.
  • Groups of rhesus macaques were immunized with 2 xlO 8 infectious units of MVA SHW-KB9 at weeks 0 and 4 by one of several routes: Tonsilar, intradermal (ID), or intramuscular (IM).
  • Tonsilar intradermal
  • IM intramuscular
  • Another group was immunized with non-recombinant MVA using the same routes.
  • Serum samples from 2 weeks after the second immunization were analyzed for binding to KB9 envelope protein by ELISA and for neutralization of SHW-89.6P and SHW-89.6.
  • soluble KB9 envelope protein was captured in 96 well plates using an antibody to the C-terminus of gpl20. Serial dilutions of sera were analyzed and used to determine the endpoint titers. Neutralization of SHW-89.6P and SHW-89.6 was determined in an MT-2 cell assay. Titers are the reciprocal serum dilution at which 50%) of the cells were protected from virus-induced killing. In in vitro neutralization assays, SHW-89.6P and SHW-89.6 are heterologous, i.e. sera from animals infected with one of the viruses do not neutralize the other virus.
  • Table E Frequencies of gag CM-9-specific CD3/CD8 T cells following immunization of macaques with MVA/SHW-KB9.
  • Groups of MamuA*01 positive rhesus macaques were immunized with 2 xlO infectious units of MVA/SHW-KB9 at weeks 0 and 4 by one of several routes: tonsilar, intradermal (ID), or intramuscular IM). Another group was immunized with non- recombinant MVA.
  • the frequencies of CD3+/CD8+ T cells that bound tetrameric complex containing the SW gag-specific peptide CM9 were determined by flow cytometry at various times after each immunization.
  • Time intervals were as follows: la, lb, and Id were one, two, and four weeks after the first immunization, respectively; 2a, 2b, 2c, and 2d were one, two, three, and twelve weeks after the second immunization, respectively. Values above background are shown in bold face. Strong S TV gag-specific responses were observed after a single immunization with MVA/SHW-KB9 in all immunized animals. Boosting was observed in most animals following the second immunization. In addition, measurable tetramer binding was still found twelve weeks after the second immunization. Table F: Frequencies of specific T cells following immunization of macaques with
  • MVA/SHW-KB9 expresses 5 genes from the chimeric virus, SHW-89.6P: envelope, gag, polymerase, tat, and nef.
  • SHW-89.6P envelope, gag, polymerase, tat, and nef.
  • the frequencies of T cells specific for each ofthe 5 antigens were analyzed using pools of peptides co ⁇ esponding to each individual protein.
  • Fresh PBMC were stimulated with pools of peptides for 30-36 hours in vitro.
  • Gamma interferon- producing cells were enumerated in an ELISPOT assay. The total number of cells specific for each antigen is given as "total # spots", hi addition, the number of responding animals and average # of spots per group is shown.
  • PBMC peripheral blood mononuclear cells
  • DNA primes were at 0 and 8 weeks and MVA/48 boost was at 24 weeks
  • Recombinant MVA vaccines have been successful in generating SW and SHW specific humoral and CDS T cell responses in non-human primates and, alone or in combination with DNA vaccines, have provided protection in rhesus macaques from disease after pathogenic SHW challenge.
  • An overall program goal is to conduct clinical vaccine trials in Africa using vaccines that induce both neutralizing antibody and CD8 T cell specific responses and that are based upon representative full-length HW-l sequences isolated from the target vaccine cohorts.
  • the predominant incident and prevalent HW-l subtype in Kenya is subtype D.
  • Several R5 subtype D HW-l strains were selected and used to prepare recombinant MVA vaccines expressing env, gag, protease and RT.
  • rMVA-UGD recombinant MVA expressing subtype D env and gag/pol
  • rMVA-UGD recombinant MVA expressing subtype D env and gag/pol were prepared in primary CEF cells using gamma-irradiated FBS from a USD A approved source and selected using GFP expression. These rMVA-UGD were further plaque-purified and amplified to titers sufficient for in-vivo immunogenicity studies. Pre-clinical humoral and cellular immunogenicity ofthe various rMVA-UGD were then assessed in BALB/c mice.
  • MVA Expressing Altered HIV-1 Envelope, Gag, and Polymerase Genes from
  • Env and gagpol genes were PCR amplified from Kenyan HW-l clade D isolates by short term co-cultures on normal human PBMC (Harris et al. 2002 AIDS Research and Human Retroviruses 18:1281) using the oligonucleotides shown in Table G and cloned into pCR2.1-TOPO (Invitrogen).
  • HW-I infected individuals contain a population or quasi-species of related but distinct viruses.
  • the resulting amplified env genes have a C-terminal deletion of 115 amino acids that was previously shown to enhance expression and yield a more stable recombinant virus.
  • the resulting gagpol genes have a deletion of the entire integrase and Rnase H portions of the genes.
  • several mutations were made by site-directed mutagenesis (Quik Change from Stratagene). In the env genes, silent mutations were made to eliminate two naturally occurring vaccinia virus early transcription termination signals (TTTTTNT, SEQ ID NO: 14) (Earl et.
  • BS-C-1 cells were infected with vTF7-3 (Fuerst et al 1986 PNAS USA 83:8122), a recombinant vaccinia virus expressing T7 RNA polymerase, transfected with a plasmid, and metabohcally labeled.
  • Cell lysates were subjected to immunoprecipitation with serum pooled from several HW-l clade D-infected individuals. Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. One env and one gagpol DNA clone from each clade D isolate was chosen for construction of recombinant MVA viruses. DNA sequencing was performed to confirm the integrity of each gene. Sequences are presented in Appendix 1.
  • MVA shuttle plasmids Two MVA shuttle plasmids, pLAS-T and pLAS-2 ( Figure 18), were used for construction of recombinant MVA viruses.
  • DNA sequences of both plasmids are presented in Appendix 2.
  • both plasmids the foreign gene is driven by the modified H5 promoter.
  • both plasmids contain a cassette with the gene for green fluorescent protein (GFP) driven by the vaccinia pi 1 promoter. This cassette is flanked by direct repeats that will readily recombine to eliminate GFP during virus propagation.
  • GFP is used as a positive screening marker in early rounds of plaque purification, and as a negative screening marker in final recombinant virus selection (Figure 22).
  • MVA flanking sequences in pLAS-1 and pLAS-2 direct recombination into deletion III (Del III) and deletion II (Del II) of MVA, respectively.
  • Gagpol genes from 2 isolates were cloned separately into pLAS-1 for insertion into Del III of MVA.
  • the plasmids were named pLAS-1/UGD/Bgag and pLAS-1/UGD/Cgag ( Figure 19 & Table I).
  • a short open reading frame precedes the env open reading frame. This open reading frame is out of frame with env and terminates at approximately nucleotide 75 in the env gene.
  • Env genes from three isolates were cloned separately into MVA shuttle plasmid pLAS-2, for insertion into Del II of MVA. Plasmids were named pLAS-2/UGD/Benv, pLAS-2/UGD/Cenv, and pLAS-2/UGD/Denv ( Figure 20 & Table I).
  • a short open reading frame precedes the env open reading frame. This open reading frame is out of frame with env and terminates at approximately nucleotide 75 in the env gene.
  • MVA 1974/NIH Clone 1 was used as the parent for all recombinant viruses. It was derived from a stock of MVA at passage 572, prepared on 2/22/1974 in the laboratory of A. Mayr in Germany. After receipt in the Laboratory of Viral Diseases, this stock was passaged a total of 6 times in chicken embryo fibroblast (CEF) cells, including 3 clonal purifications. Amplification was performed on the final, clonally purified virus. All CEF cells were derived from specific pathogen-free (SPAFAS) eggs.
  • CEF chicken embryo fibroblast
  • Recombinant viruses expressing gagpol CEF cells were infected with MVA 1974/NIH Clone 1 and transfected with either pLAS-1/UGD/Bgag or pLAS-1/UGD/Cgag for insertion into Del HI. Two to three rounds of plaque purification were performed based on GFP expression. Further rounds of plaque purification were performed by picking plaques based on lack of GFP expression and concomitant positive gag expression as measured by immunostaining using a monoclonal antibody to HW-l p24 (183-H12-5C; obtained from the NIH AIDS Research and Reference Reagent Program) ( Figure 22). Recombinant gagpol-expressing viruses were amplified and characterized for gag expression by immunoprecipitation as described above. The two viruses were named MVA/UGD/Bgag and MVA/UGD/Cgag. These viruses were then used as the parent in making gagpol/env recombinant viruses (see below).
  • Recombinant viruses expressing gagpol and env Recombinant viruses, MVA/UGD/Bgag and MVA/UGD/Cgag were used as parent viruses for insertion of e/?v genes.
  • CEF cells were infected with either MVA/UGD/Bgag or MVA/UGD/Cgag and subsequently transfected with one of the pLAS-2-env-containing plasmids described above ( Figure 23 & Table I).
  • the first two rounds of plaque purification were performed based on GFP expression, hi subsequent rounds of purification, plaques were selected based on loss of GFP expression and positive gag and env expression as measured by immunostaining in duplicate cultures ( Figure 22).
  • a total of 5 gagpol/env-expressing viruses (MVA/UGD-1 through -5) were amplified and characterized (Table J). Characterization of recombinant MVA/UGD viruses: The 5 MVA/UGD viruses have been characterized for gene expression and function. Immunoprecipitation of env and gag proteins is shown in Figure 24. BS-C-1 cells were infected with individual recombinant viruses at a multiplicity of infection of 10, metabohcally labeled, and lysates were subjected to immunoprecipitation with a pool of sera from HW-l clade D infected individuals.
  • Viruses expressing gagpol only (MVA UGD/Bgag and Cgag) were included, as was non-recombinant MVA as a negative control and MVA/CMDR as a positive control. The latter virus expresses gagpol/env from a Clade E HW-l isolate. All viruses produced high levels of gag protein and efficient processing into p24 was observed, hi addition, all env-expressing viruses produced high levels of env protein (gp 160).
  • Figure 25 demonstrates that the gag and env proteins produced by the MVA/UGD viruses are functional.
  • Virus-like particles were obtained by centrifugation of the supernatant of infected cells through a sucrose cushion (Karacostas et al. 1993 Virology 193:661). Pelleted material was then separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography (Panel A). As seen, p55 and p24 gag proteins were found in the pellet indicating that virus-like particles were formed.
  • Panel B shows results of an assay in which env-expressing cells (infected with MVA/UGD virus) were mixed with cells expressing CD4 and co-receptor (X4 or R5) (Nussbaum, Broder, & Berger 1994 J. Virol 68:5411). Fusion was measured by beta-galactosidase activity in cell lysates. As shown, all five MVA/UGD viruses induced fusion with CD4/R5-expressing cells.
  • Immunogenicity of recombinant MVA UGD viruses (study 1): Groups of Balb/c mice were immunized with individual MVA/UGD viruses, non- recombinant MVA (negative control), or MVA/CMDR (positive control - expressing clade E gagpol/env) at weeks 0 and 3. The dose was 10 7 infectious units per immunization and the route was intraperitoneal. Humoral and cell mediated responses were measured and are shown in Figures 26-28.
  • Antibody responses after 2 immunizations are shown in Figure 26.
  • Reciprocal endpoint ELISA titers to p24 at various times after immunization are shown in Panel A. All UGD viruses elicited gag-specific antibodies after 2 immunizations. Env-specific responses are shown in Panel B. hi this experiment, pooled sera from groups of mice were used to immunoprecipitate metabohcally labeled, autologous gpl 60 proteins. As seen, sera from mice immunized with MVA/UGD- 1, -3, and -4 reacted with gpl 60 (the other viruses were not tested in this assay). Reciprocal endpoint titers to gpl 40 env at various times after immunization are shown in Panel C All UGD viruses elicited env-specific antibodies after 2 immunizations.
  • T cell responses were measured with several assays.
  • gag and pol peptide-specific intracellular interferon gamma (IFN- ⁇ ) responses were measured by intracellular cytokine staining.
  • Splenocytes were collected 3 weeks after immunization, stimulated in- vitro for 7 days, and then cultured overnight with peptide-pulsed P815 cells.
  • Brefeldin A was added to prevent secretion of INF- ⁇ .
  • CD3 positive, CD8 positive, IFN- ⁇ positive cells were enumerated by flow cytometry. Analyses were performed after one and two immunizations.
  • gag- and pol-specific responses were observed after two immunizations (Figure 27) (samples from animals immunized with MVA/UGD-2, and 5 were not assayed).
  • gag- and pol-specific INF- ⁇ responses were measured by ELISPOT ( Figure 28 A & B). Briefly, splenocytes from immunized mice were mixed with gag or pol peptide-pulsed P815 cells in 96-well nitrocellulose plates coated with anti-IFN- ⁇ antibody. After overnight incubation, spots were visualized by sequential incubation with anti-IFN- ⁇ biotin antibody, stiaptavidin-HRP, and AEC substrate.
  • Gag peptide-specific responses were found after one immunization and were boosted in most groups after the second immunization. Pol peptide-specific responses were found in several groups after two immunizations. Third, gag peptide-specific responses were measured by tetramer staining (H-2Kd gag LAI tetiamer: AMQMLKETI, SEQ ID NO: 63) ( Figure 29). Splenocytes were stimulated in vitro with either gag peptide or MVA/CM240gagpol, a recombinant virus expressing a clade E gagpol.
  • CD3 positive, CD8 positive, tetramer positive cells were enumerated by flow cytometry. Positive tetramer staining was observed with cells from several groups of mice. Immunogenieity of recombinant MVA UGD viruses (study 2):
  • mice were administered intraperitoneal immunizations of 10 7 infectious units of MVA at weeks 0 and 3. Five mice per group were sacrificed two weeks after the 1 st and 2 nd immunizations and spleens were removed for assessment of cellular immunogenicity. Sera were collected from each mouse at weeks -1, 0, 1, 2, 3, 4 and 5. Splenocytes and sera were pooled together by group. HW gag-specific serum IgG responses were detected from all MVA/UGD-irnmunized groups after the 2 nd immunization ( Figure 30). These gag-specific responses were predominantly of subclass IgG2a for both MVA/UGD-3 and MVA/UGD-4 demonstrating a Thl -type response (Table Q).
  • HW-specific cell-mediated immunity was assessed by four separate assays: (1) intracellular IFN- ⁇ staining by flow cytometry (ICS), (2) IFN- ⁇ secretion by ELISPOT, (3) gag-peptide specific tetramer staining and (4) cytotoxic T lymphocyte (CTL) killing.
  • ICS flow cytometry
  • IFN- ⁇ secretion by ELISPOT IFN- ⁇ secretion by ELISPOT
  • gag-peptide specific tetramer staining (4) cytotoxic T lymphocyte (CTL) killing.
  • CTL cytotoxic T lymphocyte
  • IFN- ⁇ ELISPOT HW gag- specific IFN- ⁇ responses were detected by ELISPOT without prior in-vitro stimulation after both the 1 st ( Figure 32A) and 2 nd immunization ( Figure 32B) with a boost detected after the 2 nd immunization. HW gag-specific responses were stronger than the pol-specific responses. HW pol-specific responses were detectable after a 7-day in-vitro stimulation with P815 cells pulsed with pol peptide ( Figure 32C).
  • Tetramer staining HIV gag peptide-specific responses were measured by tetramer staining (H-2Kd gag LAI tetramer: AMQMLKETI, SEQ ID NO: 63) ( Figure 33).
  • Splenocytes were stained pre or post a 7- day in-vitro stimulation with P815 cells either pulsed with gag peptide or infected with MVA/CM240gag/pol, a recombinant virus expressing a subtype E gagpol.
  • CD3 positive/CD8 positive/tetramer positive cells were enumerated by flow cytometry. Positive tetramer staining was observed for all of the MVA/UGD immunized groups both pre-WS and post-WS with MVA-infected P815 cells.
  • HW- 1 sequences from Kenya were selected.
  • the predominant incident and prevalent HW-l subtype in Kenya is subtype A.
  • Several R5 subtype A HW-l strains were selected and used to prepare recombinant MVA vaccines expressing env and gagpol (gag, protease and RT).
  • gagpol gage-binding protein
  • One gagpol and two env clones from pure Kenyan subtype A isolates were selected.
  • Site directed mutagenesis in env to eliminate vaccinia virus early transcription termination sites Site directed mutagenesis in pol to inactivate enzymatic activity.
  • gagpol into MVA 1974/NIH Clone 1 using primary CEF cells.
  • the MVA/KEA viruses were characterized for gene expression and function, hmnunoprecipitation of env and gag protiens is shown in Figure 35.
  • BSC-1 cells were infected with individual recombinant viruses at a multiplicity of infection of 10,
  • MVA/UGD and WR/vEJW-1 were included as positive controls for env and gag expression, respectively. All MVA/KEA viruses express high levels of env and/or gag, as expected.
  • Virus-like particles were obtained by centrifugation of the supernatant of infected cells through a sucrose cushion (Karacostas et al. 1993 Virology 193:661), Gag p24 protein was found in the pelleted material indicating the formation of virus-like particles ( Figure 36).
  • a cell-cell fusion assay was used to assess the function of expressed, membrane
  • the 5 recombinant MVA viruses expressing env, gag and pol from Kenyan subtype A HW-l isolates (MVA/KEA- 1 through MVA/KEA-5), the 3 viruses expressing subtype A env alone (MVA/KEA-6 through MVA/KEA-8) and the MVA expressing subtype A gag/pol (MVA/KEA-9) were evaluated in an in-vivo mouse immunogenicity study
  • mice 25 designed to measure the humoral and cellular immunogenicity of these vaccines.
  • BALB/c mice (10 per group) were administered intraperitoneal immunizations of 10 7 infectious units of individual MVA/KEA viruses at weeks 0 and 3.
  • Five mice per group were sacrificed two weeks after the 1 st and 2 nd immunizations and spleens were removed for assessment of cellular immunogenicity.
  • Sera were collected from each mouse at weeks — 1,
  • HW env-specific serum IgG responses were detected from all MVA/KEA-immunized groups after the 2 nd immunization ( Figure 38). While env-specific responses were detected in all groups except for the contiol group, they were strongest in MVA/KEA-3, 4, 5, 6 and 8. HW-specific cell-mediated immunity was assessed by three assays: (1) intracellular IFN- ⁇ staining by flow cytometry (ICS), (2) IFN- ⁇ secretion by ELISPOT, and (3) gag- peptide specific tetramer staining.
  • ICS flow cytometry
  • IFN- ⁇ secretion by ELISPOT IFN- ⁇ secretion by ELISPOT
  • ICS Splenocytes were collected two weeks after the 2 nd immunization, stimulated for 7 days with MVA-infected P815 cells and then incubated overnight with P815 cells pulsed with a gag or pol peptide previously shown to be target of CD8 T cells in BALB/c mice (Casimiro et al. 2002 J Virol 76:185). Brefeldin A was included during the overnight incubation to prevent cytokine secretion.
  • HW gag-specific responses were detected for each of the groups immunized with MVA/KEA viruses expressing env, gag and pol or gag and pol, but not control immunized, mice as evidenced by the production of intracellular IFN- ⁇ after peptide stimulation ( Figure 39).
  • Splenocytes positive for HW gag ranged from 1% to 22% for the MVA/KEA-immunized mice.
  • IFN- ⁇ ELISPOT HW gag-specific IFN- ⁇ responses from splenocytes without prior in- vitro stimulation were detected in all groups receiving MVA/KEA viruses expressing gag after the 1 st immunization ( Figure 40).
  • Gag, and Pol for Use in Africa HIV- 1 sequences from Africa were selected.
  • the predominant incident and prevalent HW-l subtype in Africa is subtype C.
  • Several R5 subtype C HW-l strains were selected and used to prepare recombinant MVA vaccines expressing env, gag, protease and RT.
  • One gagpol and one env clone from pure African subtype C isolates were selected.
  • Steps described in EXAMPLE 5 were followed for the selected subtype C clones. These include: PCR amplification of truncated genes and cloning into pCR2.1.
  • the KEA viruses were characterized for gene expression. Immunoprecipitation of env and gag proteins is shown in Figure 42. BSC-1 cells were infected with individual recombinant viruses at a multiplicity of infection of 10, metabohcally labeled, and lysates were subjected to immunoprecipitation with a pool of sera from HW-l infected individuals. MVA/CMDR and MVA were included as positive and negative controls, respectively. The MVA/KEA virus expresses high levels of env and gag.
  • Virus-like particles were obtained by centrifugation of the supernatant of infected cells through a sucrose cushion (Karacostas et al. 1993 Virology 193:661). Gag p24 protein was found in the pelleted material indicating the formation of virus-like particles ( Figure 43).
  • Stability of expression of HIV-1 genes in recombinant MVA viruses The stability of the inserted env and gagpol genes in recombinant viruses from each of the subtypes was tested after serial passage in CEF cells. Viruses were grown in CEF cells using a procedure that mimics that used for expansion of virus for large scale vaccine production, i.e. infection at low multiplicity, growth for 3 days at 37C, harvesting of virus from cell lysates. After repeated passage, the virus stocks were tested for stability of the inserts using a 3 -day immunostaining protocol. In this protocol, CEF cell monolayers were infected at a low multiplicity allowing for visualization of individual viral foci.
  • Appendix 1 DNA sequences of gagpol and env genes from Kenyan HIV-1 clade D isolates:
  • KER2008 gagpol (SEQ ID NO: 58):
  • TZA-246 gagpol (SEQ ID NO: 61):

Abstract

The invention provides modified virus Ankara (MVA), a replication-deficient strain of vaccinia virus, expressing human immunodeficiency virus (HIV) env, gag, and pol genes.

Description

MVA EXPRESSING MODIFIED HIV ENVELOPE, GAG, AND POL GENES
Field ofthe Invention
The invention provides modified vaccinia Ankara (MVA), a replication-deficient strain of vaccinia vims, expressing human immunodeficiency vims (HIV) env, gag, and pol genes.
Background ofthe Invention
Cellular immunity plays an important role in the control of immunodeficiency vims infections (P J. Goulder et al. 1999 AIDS 13:S121). Recently, a DNA vaccine designed to enhance cellular immunity by cytokine augmentation successfully contained a highly virulent immunodeficiency virus challenge (D.H. Barouch et al. 2000 Science 290:486).
Another promising approach to raising cellular immunity is DNA priming followed by recombinant poxvirus boosters (H.L. Robinson et al. 2000 AIDS Rev 2:105). This heterologous prime/boost regimen induces 10- to 100-fold higher frequencies of T cells than priming and boosting with DNA or recombinant poxvirus vaccines alone. Previously, investigators showed that boosting a DNA-primed response with a poxvirus was superior to boosting with DNA or protein for the control of a non-pathogenic immunodeficiency virus
(H.L. Robinson et al. 1999 Nat Med 5:526). There is a need for the control of a pathogenic immunodeficiency vims. Summary ofthe Invention
Here we report that DNA priming followed by a recombinant modified vaccinia
Ankara (rMVA) booster has controlled a highly pathogenic immunodeficiency vims challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency vims proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered seven months after the booster. These findings are envisioned as indicating that a relatively simple multiprotein DNA/MVA vaccine .can help to control the acquired immune deficiency syndrome (AIDS) epidemic. We also report that inoculations of rMVA induce good immune responses even without DNA priming. Brief Description ofthe Drawings
Figure 1. Phylo genetic relationships of HIV- 1 and HIV-2 based on identity of pol gene sequences. SrVcpz and SlVsmm are subhuman primate lentiviruses recovered from a chimpanzee and sooty mangabey monkey, respectively. Figure 2. Phylogenetic relationships of HIV-1 groups M, N and O with four different SrVcpz isolates based on full-length pol gene sequences. The bar indicates a genetic distance of 0.1 (10% nucleotide divergence) and the asterisk positions group N HIV-1 isolates based on env sequences. Figure 3. Tropic and biologic properties of HIV-1 isolates.
Figure 4. HTV-encoded proteins. The location of the HTV genes, the sizes of primary translation products (in some cases polyproteins), and the processed mature viral proteins are indicated.
Figure 5. Schematic representation of a mature HIV-1 virion. Figure 6. Linear representation of the HIV-1 Env glycoprotein. The arrow indicates the site of gpl60 cleavage to gpl20 and gp41. hi gpl20, cross-hatched areas represent variable domains (Vi to V5) and open boxes depict conserved sequences (Ci to C5). In the gp41 ectodomain, several domains are indicated: the N-terminal fusion peptide, and the two ectodomain helices (N- and C-helix). The membrane-spanning domain is represented by a black box. In the gp41 cytoplasmic domain, the Tyr-X-X-Leu (YXXL) endocytosis motif (SEQ ID NO: 9) and two predicted helical domains (helix- 1 and -2) are shown. Amino acid numbers are indicated.
Figure 7. Temporal frequencies of Gag-specific T cells. (A) Gag-specific CD8 T cell responses raised by DNA priming and rMVA booster immunizations. The schematic presents mean Gag-CM9-tetramer data generated in the high-dose i.d. DNA-immunized animals. (B) Gag-specific IFN-γ ELISPOTs in A*01 (open bars) and non-A*01 (filled bars) macaques at various times before challenge and at two weeks after challenge. Three pools of 10 to 13 Gag peptides (22-mers overlapping by 12) were used for the analyses. The numbers above data bars represent the arithmetic mean ± SD for the ELISPOTs within each group. The numbers at the top ofthe graphs designate individual animals. *, data not available; #, <20 ELISPOTs per lxl 06 peripheral blood mononuclear cells (PBMC). Temporal data for Gag-CM9-Mamu-A*01 tetramer-specific T cells can be found in Figure 12.
Figure 8. Temporal viral loads, CD4 counts, and survival after challenge of vaccinated and control animals. (A) Geometric mean viral loads and (B) geometric mean CD4 counts. (C) Survival curve for vaccinated and control animals. The dotted line represents all 24 vaccinated animals. (D) Viral loads and (E) CD4 counts for individual animals in the vaccine and control groups. The key to animal numbers is presented in (E). Assays for the first 12 weeks after challenge had a detection level of 1000 copies of RNA per milliliter of plasma. Animals with loads below 1000 were scored with a load of 500. For weeks 16 and 20, the detection level was 300 copies of RNA per milliliter. Animals with levels of virus below 300 were scored at 300. Figure 9. Postchallenge T cell responses in vaccine and control groups. (A)
Temporal tetramer+ cells (dashed line) and viral loads (solid line). (B) Ihtiacellular cytokine assays for IFN-γ production in response to stimulation with the Gag-CM9 peptide at two weeks after challenge. This ex vivo assay allows evaluation of the functional status of the peak postchallenge tetramer"1" cells displayed in Figure 7A. (C) Proliferation assay at 12 weeks after challenge. Gag-Pol-Env (open bars) and Gag-Pol (hatched bars) produced by transient tiansfections were used for stimulation. Supernatants from mock-transfected cultures served as control antigen. Stimulation indices are the growth of cultures in the presence of viral antigens divided by the growth of cultures in the presence of mock antigen. Figure 10. Lymph node histomorpho logy at 12 weeks after challenge. (A) Typical lymph node from a vaccinated macaque showing evidence of follicular hyperplasia characterized by the presence of numerous secondary follicles with expanded germinal centers and discrete dark and light zones. (B) Typical lymph node from an infected contiol animal showing follicular depletion and paracortical lymphocellular atrophy. (C) A representative lymph node from an age-matched, uninfected macaque displaying nonreactive germinal centers. (D) The percentage ofthe total lymph node area occupied by germinal centers was measured to give a non-specific indicator of follicular hyperplasia. Data for uninfected controls are for four age-matched rhesus macaques.
Figure 11. Temporal antibody responses. Micrograms of total Gag (A) or Env (B) antibody were determined with ELISAs. The titers of neutralizing antibody for SHTV-89.6 (C) and SHIV-89.6P (D) were determined with MT-2 cell killing and neutral red staining (D.C. Montefiori et al. 1988 J Gin Microbiol 26:231). Titers are the reciprocal of the serum dilution giving 50% neutralization of the indicated viruses grown in human PBMC. Symbols for animals are the same as in Figure 8. Figure 12. Gag-CM9-Mamu-A*01 tetramer-specific T cells in Mamu-A*01 vaccinated and contiol macaques at various times before challenge and at two weeks after challenge. The number at the upper right comer of each plot represents the frequency of tetramer-specific CD8 T cells as a % of total CD8 T cells. The numbers above each column of F ACS data designate individual animals.
Figure 13. Map of plasmid transfer vector pLW-48.
Figure 14 A-I. Sequences of plasmid transfer vector pLW-48, Psy II promoter (which controls ADA envelope expression), ADA envelope truncated, PmH5 promoter (which controls HXB2 gag pol expression), and HXB2 gag pol (with safety mutations, Δ integrase).
Figure 15. Plasmid transfer vector pLW-48 and making MVA recombinant virus MVA/HIV 48. Figure 16. A clade B gag pol.
Figure 17. Sequence of new Psyn II promoter. Figure 18. ρLAS-1 and ρLAS-2. Figure 19. ρLAS-1/UGDgag. Figure 20. ρLAS-2/XJGDenv. Figure 21. pLAS-2/UGDrev env.
Figure 22. Schematic for recombinant MVA production. Figure 23. Overview of making recombinant MVA/UGD viruses. Figure 24. Immunoprecipitation analysis.
Figure 25. Functional analysis of expressed proteins. A, Virus-like partikle assay. B. Env fusion assay.
Figure 26. MVA/UGD induced HIV env-and gag-specific antibody responses. A. HIV p24-specofoc serum IgG responses. B. HIV env-specific serum IgG responses. C. MVA UGD induced HIV env- and gag-specific antibody responses (study 1).
Figure 27. JVJVA/UGD induced gag-specific intracellular IFN-γ production. Figure 28A. MVA/UGD induced gag-specific IFN-γ ELISPOT.
Figure 28B. MVA/UGD induced pol-specific IFN-γ ELISPOT. Figure 29. MVA/UGD induced gag-specific tetramer staining. Figure 30. MVA/UGD induced gag-specific antibody responses (study 2). Figure 31A & B. MVA/UGD induced gag-and pol-specific intracellular IFN-γ production (study 2).
Figure 32 A, B, & C. MVA/UGD induced gag-and pol-specific IFN.-γ ELISPOT (study 2).
Figure 33. MVA/UGD induced gag-specific tetramer staining (study 2). Figure 34. JVTVA/UGD induced gag-specific cytotoxic T cell killing. Figure 35. Immunoprecipitation analysis of cell lysates (MVA/KEA). Figure 36. Gag particle assay (MVA/KEA). Figure 37. Fusion assay (MVA/KEA). Figure 38. MVA/KEA induced HIV-1 env-specific antibody responses.
Figure 39. MVA/KEA induced gag-specific intracellular IFN-γ production. Figure 40. MVA/KEA induced gag-specific tetramer staining. Figure 41. MVA/KEA induced gag-specific IFN-γ ELISPOT. Figure 42. Immunoprecipitation analysis of cell lysates (MVA/TZC). Figure 43. Fusion assay (MVA/TZC).
Brief Description ofthe Appendices Appendix 1. DNA sequences of gagpol and env genes from Ugandan HIV-1 clade D isolates.
Appendix 2. DNA sequences of MVA shuttle plasmids, pLAS-1 and pLAS-2. Appendix 3. DNA sequences of gagpol and env genes from Kenyan HIV-1 clade
A isolates.
Appendix 4. DNA sequences of gagpol and env genes from Tanzanian HIV-1 clade C isolates.
Appendix 5. American Type Culture Collection, Budapest Treaty Deposit Form BP/1 , questions 1 -8, regarding MVA 1974/NIH Clone 1.
Appendix 6. American Tissue Type Collection, Additional Information Required When Depositing A Virus for Patent Purposes, regarding MVA 1974/NIH Clone 1.
Deposit of Microorganism The following microorganism has been deposited in accordance with the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), Manassas, VA, on the date indicated:
MVA 1974/NIH Clone 1 was deposited as ATCC Accession No. PTA-5095 on March 27, 2003 with the American Type Culture Collection (ATCC), 10801 University
Blvd., Manassas, VA 20110-2209, USA. This deposit was made under the provisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicant and ATCC which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability ofthe progeny to one detennined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC § 122 and the Commissioner's rules pursuant thereto (including 37 CFR § 1.14). Availability of the deposited strain is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
Detailed Description ofthe Prefeπed Embodiment Recombinant MVA Virus
Vaccinia virus, a member of the genus Orthopoxvirus in the family of Poxviridae, was used as live vaccine to immunize against the human smallpox disease. Successful worldwide vaccination with vaccinia virus culminated in the eradication of variola virus, the causative agent of the smallpox ("The global eradication of smallpox. Final report of the global commission for the certification of smallpox eradication". History of Public Health, No. 4, Geneva: World Health Organization, 1980). Since that WHO declaration, vaccination has been universally discontinued except for people at high risk of poxvirus infections (e.g. laboratory workers).
More recently, vaccinia viruses have also been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M. et al. 1982 PNAS USA 79:7415-7419; Smith, G.L. et al. 1984 Biotech Genet Engin Rev 2:383-407). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non- essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83,286 and No. 110,385). The recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
For vector applications health risks would be lessened by the use of a highly attenuated vaccinia virus strain. Several such strains of vaccinia virus were especially developed to avoid undesired side effects of smallpox vaccination. Thus, the modified vaccinia Ankara (MVA) has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A. et al. 1975 Infection 3:6-14; Swiss Patent No. 568,392). The MVA virus is publicly available from American Type Culture Collection as ATCC No. VR-1508. MVA is distinguished by its great attenuation, that is to say by dimimshed virulence and ability to replicate in primate cells while maintaining good immunogenicity. The MVA virus has been analyzed to deteπnine alterations in the genome relative to the parental CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). The resulting MVA virus became severely host cell restricted to avian cells.
Furthermore, MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr A. et al. 1978 Zentralbl Bakteriol [B] 167:375-390; Stick! et al 1974 Dtsch Med Wschr 99:2386-2392). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine.
MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. 1992 PNAS USA 89:10847-10851). Additionally, novel vaccinia vector vaccines were established on the basis of MVA having foreign DNA sequences inserted at the site of deletion III within the MVA genome (Sutter, G. et al 1994 Vaccine 12:1032- 1040).
The recombinant MVA vaccinia viruses can be prepared as set out hereinafter. A DNA-construct which contains a DNA-sequence which codes for a foreign polypeptide flanked by MVA DNA sequences adjacent to a naturally occurring deletion, e.g. deletion III, or other non-essential sites, within the MVA genome, is introduced into cells infected with MVA, to allow homologous recombination. Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker. The DNA-construct to be inserted can be linear or circular. A plasmid or polymerase chain reaction product is preferred. The DNA-construct contains sequences flanking the left and the right side of a naturally occuπing deletion, e.g. deletion III, within the MVA genome. The foreign DNA sequence is inserted between the sequences flanking the naturally occuπing deletion. For the expression of a DNA sequence or gene, it is necessary for regulatory sequences, which are required for the transcription of the gene, to be present on the DNA. Such regulatory sequences (called promoters) are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385). The DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al. 1973 Virol 52:456- 467; Wigler el al. 1979 Cell 16:777-785), by means of electroporation (Neumann et al. 1982 EMBO J 1:841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101:482-492), by means of liposomes (Stiaubinger et al. 1983 Meth Enzymol 101:512- 527), by means of spheroplasts (Schaffner 1980 PNAS USA 77:2163-2167) or by other methods known to those skilled in the art. HIVs and Their Replication
The etiological agent of acquired immune deficiency syndrome (AIDS) is recognized to be a retrovirus exhibiting characteristics typical of the lentivirus genus, refeπed to as human immunodeficiency virus (HIV). The phylogenetic relationships ofthe human lentiviruses are shown in Figure 1. HIV-2 is more closely related to SIVsmm, a virus isolated from sooty mangabey monkeys in the wild, than to HIV-1. It is currently believed that HIV-2 represents a zoonotic tiansmission of SrVsram to man. A series of lentiviral isolates from captive chimpanzees, designated SIVCpZ, are close genetic relatives ofHrV-1. The earliest phylogenetic analyses of HIV-1 isolates focused on samples from
Europe/North America and Africa; discrete clusters of viruses were identified from these two areas of the world. Distinct genetic subtypes or clades of HIV-l were subsequently defined and classified into three groups: M (major); O (outlier); and N (non-M or O) (Fig. 2). The M group of HTV-1, which includes over 95% ofthe global virus isolates, consists of at least eight discrete clades (A, B, C, D, F, G, H, and J), based on the sequence of complete viral genomes. Members of HIV-l group O have been recovered from individuals living in Cameroon, Gabon, and Equatorial Guinea; their genomes share less than 50% identity in nucleotide sequence with group M viruses. The more recently discovered group N HTV-1 strains have been identified in infected Cameroonians, fail to react sero logically in standard whole-virus enzyme-linked immunosorbent assay (ELISA), yet are readily detectable by conventional Western blot analysis.
Most current knowledge about HIV-1 genetic variation comes from studies of group M viruses of diverse geographic origin. Data collected during the past decade indicate that the HIV-1 population present within an infected individual can vary from 6% to 10% in nucleotide sequence. HIV-1 isolates within a clade may exhibit nucleotide distances of 15% in gag and up to 30% in gpl20 coding sequences. Interclade genetic variation may range between 30% and 40% depending on the gene analyzed. All of the H -l group M subtypes can be found in Africa. Clade A viruses are genetically the most divergent and were the most common HW-l subtype in Africa early in the epidemic. With the rapid spread of HTV-1 to southern Africa during the mid to late 1990s, clade C viruses have become the dominant subtype and now account for 48% of HW-l infections worldwide. Clade B viruses, the most intensively studied HW-l subtype, remain the most prevalent isolates in Europe and North America.
High rates of genetic recombination are a hallmark of retroviruses. It was initially believed that simultaneous infections by genetically diverse virus strains were not likely to be established in individuals at risk for HW-l. By 1995, however, it became apparent that a significant fraction of the HW-l group M global diversity included interclade viral recombinants. It is now appreciated that HW-l recombinants will be found in geographic areas such as Africa, South America, and Southeast Asia, where multiple HW-l subtypes coexist and may account for more than 10% of circulating HW-l strains. Molecularly, the genomes of these recombinant viruses resemble patchwork mosaics, with juxtaposed diverse HW-l subtype segments, reflecting the multiple crossover events contributing to their generation. Most HW-l recombinants have arisen in Africa and a majority contains segments originally derived from clade A viruses, hi Thailand, for example, the composition of the predominant circulating strain consists of a clade A gag plus pol gene segment and a clade E env gene. Because the clade E env gene in Thai HW-l strains is closely related to the clade E env present in virus isolates from the Central African Republic, it is believed that the original recombination event occuπed in Africa, with the subsequent introduction of a descendent virus into Thailand. Interestingly, no full-length HW-l subtype E isolate (i.e., with subtype E gag, pol, and env genes) has been reported to date.
The discovery that α and β chemokine receptors function as coreceptors for virus fusion and entry into susceptible CD4 cells has led to a revised classification scheme for HW-l (Fig. 3). Isolates can now be grouped on the basis of chemokine receptor utilization in fusion assays in which HW-l gpl20 and CD4+ coreceptor proteins are expressed in separate cells. As indicated in Figure 3, HW-l isolates using the CXCR4 receptor (now designated X4 viruses) are usually T cell line (TCL)-tiopic syncytium inducing (SI) strains, whereas those exclusively utilizing the CCR5 receptor (R5 viruses) are predominantly macrophage (M)-tiopic and non-syncytium inducing (NSI). The dual-tropic R5/X4 strains, which may comprise the majority of patient isolates and exhibit a continuum of tropic phenotypes, are frequently SI.
As is the case for all replication-competent retroviruses, the three primary HW-l translation products, all encoding structural proteins, are initially synthesized as polyprotein precursors, which are subsequently processed by viral or cellular proteases into mature particle-associated proteins (Fig. 4). The 55-kd Gag precursor Pr55Gag is cleaved into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. Autocatalysis ofthe 160-kd Gag-Pol polyprotein, Prl60Gag"Po1, gives rise to the protease (PR), the heterodimeric reverse transcriptase (RT), and the integrase (IN) proteins, whereas proteolytic digestion by a cellular enzyme(s) converts the glycosylated 160-kd Env precursor gpl60 to the gpl20 surface (SU) and gp41 tiansmembrane (TM) cleavage products. The remaining six HW-1- encoded proteins (Vif, Vpr, Tat, Rev, Vpu, and Nef) are the primary translation products of spliced mRNAs. Gag
The Gag proteins of HW, like those of other retroviruses, are necessary and sufficient for the formation of noninfectious, virus-like particles. Retroviral Gag proteins are generally synthesized as polyprotein precursors; the HW-l Gag precursor has been named, based on its apparent molecular mass, Pr55Gag. As noted previously, the mRNA for Pr55G g is the unspliced 9.2-kb transcript (Fig. 4) that requires Rev for its expression in the cytoplasm. When the pol ORF is present, the viral protease (PR) cleaves Pr55Gag during or shortly after budding from the cell to generate the mature Gag proteins pi 7 (MA), p24 (CA), p7 (NC), and p6 (see Fig. 4). In the virion, MA is localized immediately inside the lipid bilayer of the viral envelope, CA forms the outer portion of the cone-shaped core structure in the center of the particle, and NC is present in the core in a ribonucleoprotein complex with the viral RNA genome (Fig. 5).
The HW Pr55Gag precursor oligomerizes following its translation and is targeted to the plasma membrane, where particles of sufficient size and density to be visible by EM are assembled. Formation of virus-like particles by Pr55Gag is a self-assembly process, with critical Gag-Gag interactions taking place between multiple domains along the Gag precursor. The assembly of virus-like particles does not require the participation of genomic RNA (although the presence of nucleic acid appears to be essential), jt?σ -encoded enzymes, or Env glycoproteins, but the production of infectious virions requires the encapsidation ofthe viral RNA genome and the incorporation ofthe Env glycoproteins and the Gag-Pol polyprotein precursor Prl60Gag"Po1. Pol
Downstream of gag lies the most highly conserved region of the HW genome, the pol gene, which encodes three enzymes: PR, RT, and IN (see Fig. 4). RT and IN are required, respectively, for reverse transcription of the viral RNA genome to a double- stranded DNA copy, and for the integration of the viral DNA into the host cell chromosome. PR plays a critical role late in the life cycle by mediating the production of mature, infectious virions. The pol gene products are derived by enzymatic cleavage of a 160-kd Gag-Pol fusion protein, refeπed to as Prl60Gaε"Po1. This fusion protein is produced by ribosomal frameshifting during translation of Pr55Gag (see Fig. 4). The frame-shifting mechanism for Gag-Pol expression, also utilized by many other retroviruses, ensures that the pol-detived proteins are expressed at a low level, approximately 5% to 10% that of Gag. Like Pr55Ga , the N-terminus of Prl60Gag"Po1 is myristylated and targeted to the plasma membrane. Protease
Early pulse-chase studies performed with avian retroviruses clearly indicated that retroviral Gag proteins are initially synthesized as polyprotein precursors that are cleaved to generate smaller products. Subsequent studies demonstrated that the processing function is provided by a viral rather than a cellular enzyme, and that proteolytic digestion of the Gag and Gag-Pol precursors is essential for virus infectivity. Sequence analysis of retroviral PRs indicated that they are related to cellular "aspartic" proteases such as pepsin and renin. Like these cellular enzymes, retroviral PRs use two apposed Asp residues at the active site to coordinate a water molecule that catalyzes the hydrolysis of a peptide bond in the target protein. Unlike the cellular aspartic proteases, which function as pseudodimers (using two folds within the same molecule to generate the active site), retroviral PRs function as true dimers. X-ray crystallographic data from HW-l PR indicate that the two monomers are held together in part by a four-stranded antiparallel β-sheet derived from both N- and C- terminal ends of each monomer. The substrate-binding site is located within a cleft formed between the two monomers. Like their cellular homologs, the HW PR dimer contains flexible "flaps" that overhang the binding site and may stabilize the substrate within the cleft; the active-site Asp residues lie in the center of the dimer. Interestingly, although some limited amino acid homology is observed suπounding active-site residues, the primary sequences of retroviral PRs are highly divergent, yet their structures are remarkably similar. Reverse Transcriptase
By definition, retroviruses possess the ability to convert their single-stranded RNA genomes into double-stranded DNA during the early stages of the infection process. The enzyme that catalyzes this reaction is RT, in conjunction with its associated RNaseH activity. Retroviral RTs have three enzymatic activities: (a) RNA-directed DNA polymerization (for minus-strand DNA synthesis), (b) RNaseH activity (for the degradation ofthe tRNA primer and genomic RNA present in DNA-RNA hybrid intermediates), and (c) DNA-directed DNA polymerization (for second- or plus-strand DNA synthesis).
The mature HW-l RT holoenzyme is a heterodimer of 66 and 51 kd subunits. The 51-kd subunit (p51) is derived from the 66-kd (p66) subunit by proteolytic removal of the C-terminal 15-kd RNaseH domain of p66 by PR (see Fig. 4). The crystal structure of HW- 1 RT reveals a highly asymmetric folding in which the orientations of the p66 and p51 subunits differ substantially. The p66 subunit can be visualized as a right hand, with the polymerase active site within the palm, and a deep template-binding cleft formed by the palm, fingers, and thumb subdomains. The polymerase domain is linked to RNaseH by the connection subdomain. The active site, located in the palm, contains three critical Asp residues (110, 185, and 186) in close proximity, and two coordinated Mg2+ ions. Mutation of these Asp residues abolishes RT polymerizing activity. The orientation of the three active-site Asp residues is similar to that observed in other DNA polymerases (e.g., the Klenow fragment of E. coli DNA poll). The p51 subunit appears to be rigid and does not form a polymerizing cleft; Asp 110, 185, and 186 of this subunit are buried within the molecule. Approximately 18 base pairs of the primer-template duplex lie in the nucleic acid binding cleft, stretching from the polymerase active site to the RNaseH domain. hi the RT-primer-template-dNTP structure, the presence of a dideoxynucleotide at the 3' end of the primer allows visualization of the catalytic complex trapped just prior to attack on the incoming dNTP. Comparison with previously obtained structures suggests a model whereby the fingers close in to trap the template and dNTP prior to nucleophilic attack of the 3'-OH of the primer on the incoming dNTP. After the addition of the incoming dNTP to the growing chain, it has been proposed that the fingers adopt a more open configuration, thereby releasing the pyrophosphate and enabling RT to bind the next dNTP. The structure of the HW-l RNaseH has also been determined by x-ray crystallography; this domain displays a global folding similar to that of E. coli RNaseH. Integrase A distinguishing feature of retro virus replication is the insertion of a DNA copy of the viral genome into the host cell chromosome following reverse transcription. The integrated viral DNA (the provirus) serves as the template for the synthesis of viral RNAs and is maintained as part of the host cell genome for the lifetime of the infected cell. Retroviral mutants deficient in the ability to integrate generally fail to establish a productive infection.
The integration of viral DNA is catalyzed by integrase, a 32-kd protein generated by PR-mediated cleavage of the C-terminal portion of the HW-l Gag-Pol polyprotein (see Fig. 4).
Retroviral IN proteins are composed of three structurally and functionally distinct domains: an N-terminal, zinc-finger-containing domain, a core domain, and a relatively nonconserved C-terminal domain. Because of its low solubility, it has not yet been possible to crystallize the entire 288-amino-acid HW-l IN protein. However, the structure of all three domains has been solved independently by x-ray crystallography or NMR methods. The crystal structure of the core domain of the avian sarcoma virus IN has also been determined. The N-terminal domain (residues 1 to 55), whose structure was solved by NMR spectioscopy, is composed of four helices with a zinc coordinated by amino acids His- 12, His- 16, Cys-40, and Cys-43. The structure ofthe N-terminal domain is reminiscent of helical DNA binding proteins that contain a so-called helix-turn-helix motif; however, in the HW-l structure this motif contributes to dimer formation. Initially, poor solubility hampered efforts to solve the structure of the core domain. However, attempts at crystallography were successful when it was observed that a Phe-to-Lys change at IN residue 185 greatly increased solubility without disrupting in vitro catalytic activity. Each monomer of the HW-l IN core domain (IN residues 50 to 212) is composed of a five- stranded β-sheet flanked by helices; this structure bears striking resemblance to other polynucleotidyl transferases including RNaseH and the bacteriophage MuA transposase. Three highly conserved residues are found in analogous positions in other polynucleotidyl transferases; in HW-l IN these are Asp-64, Asp-116 and Glu-152, the so-called D,D-35-E motif. Mutations at these positions block HW IN function both in vivo and in vitro. The close proximity of these three amino acids in the crystal structure of both avian sarcoma virus and HW-l core domains supports the hypothesis that these residues play a central role in catalysis ofthe polynucleotidyl transfer reaction that is at the heart ofthe integration process. The C-terminal domain, whose structure has been solved by NMR methods, adopts a five-stranded β-barrel folding topology reminiscent of a Src homology 3 (SH3) domain. Recently, the x-ray structures of SIV and Rous sarcoma virus IN protein fragments encompassing both the core and C-terminal domains have been solved. Env
The HW Env glycoproteins play a major role in the virus life cycle. They contain the determinants that interact with the CD4 receptor and coreceptor, and they catalyze the fusion reaction between the lipid bilayer of the viral envelope and the host cell plasma membrane. In addition, the HW Env glycoproteins contain epitopes that elicit immune responses that are important from both diagnostic and vaccine development perspectives.
The HW Env glycoprotein is synthesized from the singly spliced 4.3-kb Vpu/Env bicistronic mRNA (see Fig. 4); translation occurs on ribosomes associated with the rough endoplasmic reticulum (ER). The 160-kd polyprotein precursor (gpl60) is an integral membrane protein that is anchored to cell membranes by a hydrophobic stop-transfer signal in the domain destined to be the mature TM Env glycoprotein, gp41 (Fig. 6). The gpl60 is cotranslationally glycosylated, forms disulfide bonds, and undergoes oligomerization in the ER. The predominant oligomeric form appears to be a trimer, although dimers and tetramers are also observed. The gpl60 is transported to the Golgi, where, like other retroviral envelope precursor proteins, it is proteolytically cleaved by cellular enzymes to the mature SU glycoprotein gpl20 and TM glycoprotein gp41 (see Fig. 6). The cellular enzyme responsible for cleavage of retroviral Env precursors following a highly conserved Lys/Arg-X-Lys/Arg-Arg motif is furin or a furin-like protease, although other enzymes may also catalyze gpl60 processing. Cleavage of gpl 60 is required for Env-induced fusion activity and virus infectivity. Subsequent to gpl 60 cleavage, gpl 20 and gp41 form a noncovalent association that is critical for transport of the Env complex from the Golgi to the cell surface. The gpl20-gp41 interaction is fairly weak, and a substantial amount of gpl20 is shed from the surface of Env-expressing cells.
The HW Env glycoprotein complex, in particular the SU (gpl 20) domain, is very heavily glycosylated; approximately half the molecular mass of gpl 60 is composed of oligosaccharide side chains. During transport of Env from its site of synthesis in the ER to the plasma membrane, many of the side chains are modified by the addition of complex sugars. The numerous oligosaccharide side chains form what could be imagined as a sugar cloud obscuring much of gpl20 from host immune recognition. As shown in Figure 6, gpl20 contains interspersed conserved (Q to C5) and variable (Vi to V5) domains. The Cys residues present in the gpl20s of different isolates are highly conserved and form disulfide bonds that link the first four variable regions in large loops.
A primary function of viral Env glycoproteins is to promote a membrane fusion reaction between the lipid bilayers of the viral envelope and host cell membranes. This membrane fusion event enables the viral core to gain entry into the host cell cytoplasm. A number of regions in both g l20 and gp41 have been implicated, directly or indirectly, in Env-mediated membrane fusion. Studies of the HA hemagglutinin protein of the orthomyxoviruses and the F protein of the paramyxoviruses indicated that a highly hydrophobic domain at the N-terminus of these proteins, refeπed to as the fusion peptide, plays a critical role in membrane fusion. Mutational analyses demonstrated that an analogous domain was located at the N-teπninus of the HW-l, HW-2, and SW TM glycoproteins (see Fig. 6). Nonhydrophobic substitutions within this region of gp41 greatly reduced or blocked syncytium formation and resulted in the production of noninfectious progeny virions.
C-terminal to the gp41 fusion peptide are two amphipathic helical domains (see Fig. 6) which play a central role in membrane fusion. Mutations in the N-terminal helix (refeπed to as the N-helix), which contains a Leu zipper-like heptad repeat motif, impair infectivity and membrane fusion activity, and peptides derived from these sequences exhibit potent antiviral activity in culture. The structure of the ectodomain of HW-l and SW gp41, the two helical motifs in particular, has been the focus of structural analyses in recent years. Structures were determined by x-ray crystallography or NMR spectioscopy either for fusion proteins containing the helical domains, a mixture of peptides derived from the N- and C-helices, or in the case of the SW structure, the intact gp41 ectodomain sequence from residue 27 to 149. These studies obtained fundamentally similar trimeric structures, in which the two helical domains pack in an antiparallel fashion to generate a six-helix bundle. The N-helices form a coiled-coil in the center of the bundle, with the C- helices packing into hydrophobic grooves on the outside.
In the steps leading to membrane fusion CD4 binding induces conformation changes in Env that facilitate coreceptor binding. Following the formation of a ternary gpl20/CD4/coreceptor complex, gp41 adopts a hypothetical conformation that allows the fusion peptide to insert into the target lipid bilayer. The formation of the gp41 six-helix bundle (which involves antiparallel interactions between the gp41 N- and C-helices) brings the viral and cellular membranes together and membrane fusion takes place. Use of Recombinant MVA Virus To Boost CD+8 Cell Immune Response
The present invention relates to generation of a CD8+ T cell immune response against an antigen and also eliciting an antibody response. More particularly, the present invention relates to "prime and boost" immunization regimes in which the immune response induced by administration of a priming composition is boosted by administration of a boosting composition. The present invention is based on inventors' experimental demonstration that effective boosting can be achieved using modified vaccinia Ankara (MVA) vectors, following priming with any of a variety of different types of priming compositions including recombinant MVA itself.
A major protective component of the immune response against a number of pathogens is mediated by T lymphocytes of the CD8+ type, also known as cytotoxic T lymphocytes (CTL). An important function of CD8+ cells is secretion of gamma interferon (IFNγ), and this provides a measure of CD8 T cell immune response. A second component ofthe immune response is antibody directed to the proteins ofthe pathogen.
The present invention employs MVA which, as the experiments described below show, has been found to be an effective means for providing a boost to a CD8+ T cell immune response primed to antigen using any of a variety of different priming compositions and also eliciting an antibody response. Remarkably, the experimental work described below demonstrates that use of embodiments of the present invention allows for recombinant MVA virus expressing an HW antigen to boost a CD8+ T cell immune response primed by a DNA vaccine and also eliciting an antibody response. The MVA was found to induce a CD8+ T cell response after intradermal, intramuscular or mucosal immunization. Recombinant MVA has also been shown to prime an immune response that is boosted by one or more inoculations of recombinant MVA.
Non-human primates immunized with plasmid DNA and boosted with the MVA were effectively protected against intramucosal challenge with live virus. Advantageously, the inventors found that a vaccination regime used intradermal, intramuscular or mucosal immunization for both prime and boost can be employed, constituting a general immunization regime suitable for inducing CD8+ T cells and also eliciting an antibody response, e.g. in humans.
The present invention in various aspects and embodiments employs an MVA vector encoding an HW antigen for boosting a CD8+ T cell immune response to the antigen primed by previous administration of nucleic acid encoding the antigen and also eliciting an antibody response.
A general aspect ofthe present invention provides for the use of an MVA vector for boosting a CD8+ T cell immune response to an HW antigen and also eliciting an antibody response.
One aspect of the present invention provides a method of boosting a CD8+ T cell immune response to an HW antigen in an individual, and also eliciting an antibody response, the method including provision in the individual of an MVA vector including nucleic acid encoding the antigen operably linked to regulatory sequences for production of antigen in the individual by expression from the nucleic acid, whereby a CD8+ T cell immune response to the antigen previously primed in the individual is boosted.
An immune response to an HW antigen may be primed by immunization with plasmid DNA or by infection with an infectious agent.
A further aspect of the invention provides a method of inducing a CD8+ T cell immune response to an HW antigen in an individual, and also eliciting an antibody response, the method comprising administering to the individual a priming composition comprising nucleic acid encoding the antigen and then administering a boosting composition which comprises an MVA vector including nucleic acid encoding the antigen operably linked to regulatory sequences for production of antigen in the individual by expression from the nucleic acid.
A further aspect provides for use of an MVA vector, as disclosed, in the manufacture of a medicament for administration to a mammal to boost a CD8+ T cell immune response to an HW antigen, and also eliciting an antibody response. Such a medicament is 'generally for administration following prior administration of a priming composition comprising nucleic acid encoding the antigen.
The priming composition may comprise any viral vector, such as a vaccinia virus vector such as a replication-deficient strain such as modified vaccinia Ankara (MVA) or NYVAC (Tartaglia et al. 1992 Virology 118:217-232), an avipox vector such as fowlpox or canarypox, e.g. the strain known as ALVAC (Paoletti et al. 1994 Dev Biol Stand 82:65-69), or an adenovirus vector or a vesicular stomatitis virus vector or an alphavirus vector.
The priming composition may comprise DNA encoding the antigen, such DNA preferably being in the form of a circular plasmid that is not capable of replicating in mammalian cells. Any selectable marker should not be resistance to an antibiotic used clinically, so for example Kanamycin resistance is prefeπed to Ampicillin resistance. Antigen expression should be driven by a promoter which is active in mammalian cells, for instance the cytomegalovirus immediate early (CMV IE) promoter.
In particular embodiments of the various aspects of the present invention, administration of a priming composition is followed by boosting with a boosting composition, or first and second boosting compositions, the first and second boosting compositions being the same or different from one another. Still further boosting compositions may be employed without departing from the present invention, hi one embodiment, a triple immunization regime employs DNA, then adenovirus as a first boosting composition, then MVA as a second boosting composition, optionally followed by a further (third) boosting composition or subsequent boosting administration of one or other or both of the same or different vectors. Another option is DNA then MVA then adenovirus, optionally followed by subsequent boosting administration of one or other or both ofthe same or different vectors. The antigen to be encoded in respective priming and boosting compositions
(however many boosting compositions are employed) need not be identical, but should share at least one CD8+ T cell epitope. The antigen may correspond to a complete antigen, or a fragment thereof. Peptide epitopes or artificial strings of epitopes may be employed, more efficiently cutting out unnecessary protein sequence in the antigen and encoding sequence in the vector or vectors. One or more additional epitopes may be included, for instance epitopes which are recognized by T helper cells, especially epitopes recognized in individuals of different HLA types. An HW antigen of the invention to be encoded by a recombinant MVA virus includes polypeptides having immunogenic activity elicited by an amino acid sequence of an HW Env, Gag, Pol, Vif, Vpr, Tat, Rev, Vpu, or Nef amino acid sequence as at least one CD8+ T cell epitope. This amino acid sequence substantially corresponds to at least one 10- 900 amino acid fragment and/or consensus sequence of a known HW Env or Pol; or at least one 10-450 amino acid fragment and/or consensus sequence of a lαiown HW Gag; or at least one 10-100 amino acid fragment and/or consensus sequence of a known HW Vif, Vpr, Tat, Rev, Vpu, or Nef.
Although a full length Env precursor sequence is presented for use in the present invention, Env is optionally deleted of subsequences. For example, regions of the gpl20 surface and gp41 transmembrane cleavage products can be deleted.
Although a full length Gag precursor sequence is presented for use in the present invention, Gag is optionally deleted of subsequences. For example, regions of the matrix protein (pi 7), regions of the capsid protein (p24), regions of the nucleocapsid protein (pi), and regions of p6 (the C-terminal peptide ofthe Gag polyprotein) can be deleted. Although a full length Pol precursor sequence is presented for use in the present invention, Pol is optionally deleted of subsequences. For example, regions of the protease protein (plO), regions of the reverse transcriptase protein (p66/p51), and regions of the integrase protein (p32) can be deleted.
Such an HW Env, Gag, or Pol can have overall identity of at least 50% to a known Env, Gag, or Pol protein amino acid sequence, such as 50-99% identity, or any range or value therein, while eliciting an immunogenic response against at least one strain of an HW.
Percent identify can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J Mol Biol 1970 48:443), as revised by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences. The prefeπed default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
In a preferred embodiment, an Env of the present invention is a variant form of at least one HW envelope protein. Preferably, the Env is composed of gpl 20 and the membrane-spanning and ectodomain of gp41 but lacks part or all of the cytoplasmic domain of gp41.
Known HW sequences are readily available from commercial and institutional HW sequence databases, such as GENBANK, or as published compilations, such as Myers et al eds., Human Retroviruses and AIDS, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Vol. I and II, Theoretical Biology and Biophysics, Los Alamos, N. Mex. (1993), or http://hiv-web.lanl.gov/.
Substitutions or insertions of an HW Env, Gag, or Pol to obtain an additional HW Env, Gag, or Pol, encoded by a nucleic acid for use in a recombinant MVA virus of the present invention, can include substitutions or insertions of at least one amino acid residue (e.g., 1-25 amino acids). Alternatively, at least one amino acid (e.g., 1-25 amino acids) can be deleted from an HW Env, Gag, or Pol sequence. Preferably, such substitutions, insertions or deletions are identified based on safety features, expression levels, immunogenicity and compatibility with high replication rates of MVA.
Amino acid sequence variations in an HW Env, Gag, or Pol ofthe present invention can be prepared e.g., by mutations in the DNA. Such HW Env, Gag, or Pol include, for example, deletions, insertions or substitutions of nucleotides coding for different amino acid residues within the amino acid sequence. Obviously, mutations that will be made in nucleic acid encoding an HW Env, Gag, or Pol must not place the sequence out of reading frame and preferably will not create complementary domains that could produce secondary mRNA structures.
HW Env, Gag, or Pol-encoding nucleic acid of the present invention can also be prepared by amplification or site-directed mutagenesis of nucleotides in DNA or RNA encoding an HW Env, Gag, or Pol and thereafter synthesizing or reverse transcribing the encoding DNA to produce DNA or RNA encoding an HW Env, Gag, or Pol, based on the teaching and guidance presented herein.
Recombinant MVA viruses expressing HW Env, Gag, or Pol of the present invention, include a finite set of HW Env, Gag, or Pol-encoding sequences as substitution nucleotides that can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein. For a detailed description of protein chemistry and structure, see Schulz, G.E. et al, 1978 Principles of Protein Structure, Springer-Verlag, New York, N.Y., and Creighton, T.E., 1983 Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, CA. For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al. eds. Current Protocols in Molecular Biology, Greene Publishing Assoc, New York, N.Y. 1994 at §§ A.l.l-A.1.24, and Sambrook, J. et al. 1989 Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. at Appendices C and D. Thus, one of ordinary skill in the art, given the teachings and guidance presented herein, will know how to substitute other amino acid residues in other positions of an HW env, gag, or pol DNA or RNA to obtain alternative HW Env, Gag, or Pol, including substitutional, deletional or insertional variants.
Within the MVA vector, regulatory sequences for expression ofthe encoded antigen will include a natural, modified or synthetic poxvirus promoter. By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA). "Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter. Other regulatory sequences including terminator fragments, polyadenylation sequences, marker genes and other sequences may be included as appropriate, in accordance with the knowledge and practice ofthe ordinary person skilled in the art: see, for example, Moss, B. (2001). Poxviridae: the viruses and their replication. In Fields Virology, D.M. Knipe, $μd P.M. Howley, eds. (Philadelphia, Lippincott Williams & Wilkins), pp. 2849-2883. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, 1998 Ausubel et al. eds., John Wiley & Sons.
Promoters for use in aspects and embodiments of the present invention must be compatible with poxvirus expression systems and include natural, modified and synthetic sequences.
Either or both of the priming and boosting compositions may include an adjuvant, such as granulocyte macrophage-colony stimulating factor (GM-CSF) or encoding nucleic acid therefor.
Administration of the boosting composition is generally about 1 to 6 months after administration ofthe priming composition, preferably about 1 to 3 months.
Preferably, administration of priming composition, boosting composition, or both priming and boosting compositions, is intradermal, intramuscular or mucosal immunization.
Administration of MVA vaccines may be achieved by using a needle to inject a suspension of the virus. An alternative is the use of a needleless injection device to administer a virus suspension (using, e.g., Biojector™ needleless injector) or a resuspended freeze-dried powder containing the vaccine, providing for manufacturing individually prepared doses that do not need cold storage. This would be a great advantage for a vaccine that is needed in rural areas of Africa. MVA is a virus with an excellent safety record in human immunizations. The generation of recombinant viruses can be accomplished simply, and they can be manufactured reproducibly in large quantities. Intiadermal, intramuscular or mucosal administration of recombinant MVA virus is therefore highly suitable for prophylactic or therapeutic vaccination of humans against AIDS which can be controlled by a CD8+ T cell response.
The individual may have AIDS such that delivery ofthe antigen and generation of a CD8+ T cell immune response to the antigen is of benefit or has a therapeutically beneficial effect.
Most likely, administration will have prophylactic aim to generate an immune response against HW or AIDS before infection or development of symptoms.
Components to be administered in accordance with the present invention may be formulated in pharmaceutical compositions. These compositions may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. As noted, administration is preferably intradermal, intramuscular or mucosal.
Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous, subcutaneous, intramuscular or mucosal injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, iso tonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as required. A slow-release formulation may be employed.
Following production of MVA particles and optional formulation of such particles into compositions, the particles may be administered to an individual, particularly human or other primate. Administration may be to another mammal, e.g. rodent such as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, dog or cat. Administration is preferably in a "prophylactically effective amount" or a
"therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, or in a veterinary context a veterinarian, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors lαiown to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, 1980, Osol, A. (ed.). hi one prefeπed regimen, DNA is administered at a dose of 250 μg to 2.5 mg/injection, followed by MVA at a dose of 106 to 109 infectious virus particles/injection. A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Delivery to a non-human mammal need not be for a therapeutic purpose, but may be for use in an experimental context, for instance in investigation of mechanisms of immune responses to an antigen of interest, e.g. protection against HW or AIDS.
Further aspects and embodiments of the present invention will be apparent to those of ordinary skill in the art, in view of the above disclosure and following experimental exemplification, included by way of illustration and not limitation, and with reference to the attached figures. EXAMPLE 1
Control of a Mucosal Challenge and Prevention of AIDS by a Multiprotein
DNA MVA Vaccine
Here we tested DNA priming and poxvirus boosting for the ability to protect against a highly pathogenic mucosal challenge. The 89.6 chimera of simian and human immunodeficiency viruses (SHW-89.6) was used for the construction of immunogens and its highly pathogenic derivative, SHW-89.6P, for challenge (G.B. Karlsson et al. 1997 J
Virol 71:4218). SHW-89.6 and SHW-89.6P do not generate cross-neutralizing antibody
(D.C. Montefiori et al. 1998 J Virol 72:3427) and allowed us to address the ability of vaccine-raised T cells and non-neutralizing antibodies to control an immunodeficiency virus challenge. Modified vaccinia Ankara (MVA) was used for the construction of the recombinant poxvirus. MVA has been highly effective at boosting DNA-primed CD8 T cells and enjoys the safety feature of not replicating efficiently in human or monkey cells
(H.L. Robinson et al 2000 AIDS Reviews 2:105).
To ensure a broad immune response both the DNA and recombinant MVA (rMVA) components of the vaccine expressed multiple immunodeficiency virus proteins. The DNA prime (DNA/89.6) expressed simian immunodeficiency virus (SW) Gag, Pol, Vif, Vpx, and Vpr and human immunodeficiency virus- 1 (HW-l) Env, Tat, and Rev from a single transcript (RJ. Gorelick et al. 1999 Virology 253:259; M.M. Sauter et al 1996 J Cell Biol 132:795). Molecularly cloned SHW-89.6 sequences were cloned into the vector pGA2 using
Clal and RsrII sites. This cloning deleted both long terminal repeats (LTRs) and nef. The SHW-89.6 sequences also were internally mutated for a 12-base pair region encoding the first four amino acids of the second zinc finger in nucleocapsid. This mutation renders SHW viruses noninfectious (R.J. Gorelick et al. 1999 Virology 253:259). A mutation in gp41 converted the tyrosine at position 710 to cysteine to achieve better expression of Env on the plasma membrane of DNA-expressing cells (MM. Sauter et al. 1996 J Cell Biol 132:795). pGA2 uses the CMV immediate early promoter without intron A and the bovine growth hormone polyadenylation sequence to express vaccine inserts. Vaccine DNA was produced by Althea (San Diego, CA). hi transient transfections of 293T cells, DNA/89.6 produced about 300 ng of Gag and 85 ng of Env per 1x10 cells.
The rMVA booster (MVA/89.6) expressed SW Gag, Pol, and HW-l Env under the control of vaccinia virus early/late promoters. The MVA double recombinant virus expressed both the HW 89.6 Env and the SW
239 Gag-Pol, which were inserted into deletion II and deletion III of MVA, respectively. The 89.6 Env protein was truncated for the COOH-terminal 115 amino acids of gp41. The modified H5 promoter controlled the expression of both foreign genes.
Vaccination was accomplished by priming with DNA at 0 and 8 weeks and boosting with rMVA at 24 weeks (Fig. 7A).
I.d. and i.m. DNA immunizations were delivered in phosphate-buffered saline (PBS) with a needleless jet injector (Bioject, Portland, OR) to deliver five i.d. 100-μl injections to each outer thigh for the 2.5-mg dose of DNA or one i.d. 100-μl injection to the right outer thigh for the 250-μg dose of plasmid. I.m. deliveries of DNA were done with one 0.5-ml injection of DNA in PBS to each outer thigh for the 2.5-mg dose and one 100-μl injection to the right outer thigh for the 250-μg dose. lxlO8 pfu of MVA/89.6 was administered both i.d. and i.m. with a needle. One 100-μl dose was delivered to each outer thigh for the i.d. dose and one 500-μl dose to each outer thigh for the i.m dose. Control animals received 2.5 mg ofthe pGA2 vector without vaccine insert with the Bioject device to deliver five 100-μl doses i.d. to each outer thigh. The control MVA booster immunization consisted of 2xl08 pfu of MVA without an insert delivered i.d. and i.m. as described for MVA/89.6.
Four groups of six rhesus macaques each were primed with either 2.5 mg (high- dose) or 250 μg (low-dose) of DNA by intradermal (i.d.) or intramuscular (i.m.) routes using a needleless jet injection device (Bioject, Portland, OR) (T.M. Allen et al. 2000 J Immunol 164:4968).
Young adult rhesus macaques from the Yerkes breeding colony were cared for under guidelines established by the Animal Welfare Act and the NTH "Guide for the Care and Use of Laboratory Animals" with protocols approved by the Emory University Institutional Animal Care and Use Committee. Macaques were typed for the Mamu-A*01 allele with polymerase chain reaction (PCR) analyses (M.A. Egan et al 2000 J Virol 74:7485; I. Ourmanov et al. 2000 J Virol 74:2740). Two or more animals containing at least one Mamu-A*01 allele were assigned to each group. Animal numbers are as follows: 1, RBr-5*; 2, Rfrn-5*; 3, RQf-5*; 4, RZe-5; 5, ROm-5; 6, RDm-5; 7, RAj-5*; 8, RJi-5*; 9, RA1-5*; 10, RDe-5*; 11, RAi-5; 12, RPr-5; 13, RKw-4*; 14, RWz-5*; 15, RGo-5; 16, RLp-4; 17, RWd-6; 18, RAt-5; 19, RPb-5*; 20, RIi-5*; 21, RIq-5; 22, RSp-4; 23, RSn-5; 24, RGd-6; 25, RMb-5*; 26, RGy-5*; 27, RUs-4; and 28, RPm-5. Animals with the A*01 ι allele are indicated with asterisks.
Gene gun deliveries of DNA were not used because these had primed non- protective immune responses in a 1996 - 98 trial (H.L. Robinson et al. 1999 Nat Med 5:526). The MVA/89.6 booster immunization (2xl08 plaque-forming units, pfu) was injected with a needle both i.d. and i.m. A control group included two mock immunized animals and two naive animals. The challenge was given at 7 months after the rMVA booster to test for the generation of long-term immunity. Because most HW-l infections are transmitted across mucosal surfaces, an intrarectal challenge was administered.
DΝA priming followed by rMVA boosting generated high frequencies of virus- specific T cells that peaked at one week following the rMVA booster (Fig. 7). The frequencies of T cells recognizing the Gag-CM9 epitope were assessed by means of Mamu- A*01 tetramers, and the frequencies of T cells recognizing epitopes throughout Gag were assessed with pools of overlapping peptides and an enzyme-linked immunospot (ELISPOT) assay (CA. Power et al. 1999 J Immunol Methods 227:99).
For tetramer analyses, about 1x10° peripheral blood mononuclear cells (PBMC) were surface-stained with antibodies to CD3 conjugated to fluorescein isothiocyanate (FITC) (FΝ-18; Biosource hitemational, Camarillo, CA), CD8 conjugated to peridinin chlorophyl protein (PerCP) (SKI; Becton Dickinson, San Jose, CA), and Gag-CM9 (CTPYDINQM)-Mα w-^* i tetramer (SEQ ID NO: 6) conjugated to allophycocyanin (APC), in a volume of 100 μl at 8° to 10°C for 30 min. Cells were washed twice with cold PBS containing 2% fetal bovine serum (FBS), fixed with 1% paraformaldehyde in PBS, and analyzed within 24 hrs on a FACScaliber (Becton Dickinson, San Jose, CA). Cells were initially gated on lymphocyte populations with forward scatter and side scatter and then on CD3 cells. The CD3 cells were then analyzed for CD8 and tetramer-binding cells. About 150,000 lymphocytes were acquired for each sample. Data were analyzed using Flo Jo software (Tree Star, San Carlos, CA).
For interferon-γ (IFN-γ) ELISPOTs, MULTISCREEN 96 well filtration plates (Millipore Inc. Bedford, MA) were coated overnight with antibody to human IFN-γ (Clone B27, Pharmingen, San Diego, CA) at a concentration of 2 μg/ml in sodium bicarbonate buffer (pH 9.6) at 8° to 10°C. Plates were washed two times with RPMI medium and then blocked for 1 hour with complete medium (RPMI containing 10% FBS) at 37°C. Plates were washed five more times with plain RPMI medium, and cells were seeded in duplicate in 100 μl complete medium at numbers ranging from 2xl04 to 5xl05 cells per well. Peptide pools were added to each well to a final concentration of 2 μg/ml of each peptide in a volume of 100 μl in complete medium. Cells were cultured at 37°C for about 36 hrs under 5% CO2. Plates were washed six times with wash buffer (PBS with 0.05% Tween-20) and then incubated with 1 μg of biotinylated antibody to human IFN-γ per milliliter (clone 7- 86-1; Diapharma Group, West Chester, OH) diluted in wash buffer containing 2% FBS. Plates were incubated for 2 hrs at 37°C and washed six times with wash buffer. Avidin- horseradish peroxidase (Vector Laboratories, Burlingame, CA) was added to each well and incubated for 30 to 60 min at 37°C. Plates were washed six times with wash buffer and spots were developed using stable DAB as substrate (Research Genetics, Huntsville, AL). Spots were counted with a stereo dissecting microscope. An ovalbumin peptide (SIINFEKL) (SEQ ID NO: 7) was included as a control in each analysis. Background spots for the ovalbumin peptide were generally <5 for 5x105 PBMCs. This background when normalized for lxl 06 PBMC was <10. Only ELISPOT counts of twice the background (>20) were considered significant. The frequencies of ELISPOTs are approximate because different dilutions of cells have different efficiencies of spot formation in the absence of feeder cells (CA. Power et al. 1999 J Immunol Methods 227: 99). The same dilution of cells was used for all animals at a given time point, but different dilutions were used to detect memory and acute responses.
Gag-CM9 tetramer analyses were restricted to macaques that expressed the Mamu- A*01 histocompatibility type, whereas ELISPOT responses did not depend on a specific histocompatibility type. As expected, the DNA immunizations raised low levels of memory cells that expanded to high frequencies within 1 week ofthe rMVA booster (Fig. 7 and 12). In Mamu-A*01 macaques, CD8 cells specific to the Gag-CM9 epitope expanded to frequencies as high as 19% of total CD8 T cells (Fig. 12). This peak of specific cells underwent a 10- to 100-fold contraction into the DNA/MVA memory pool (Fig. 7A and 12). ELISPOTs for three pools of Gag peptides also underwent a major expansion (frequencies up to 4000 spots for lxlO6 PBMC) before contracting from 5- to 20-fold into the DNA/MVA memory response (Fig. 7B). The frequencies of ELISPOTs were the same in macaques with and without the A *01 histocompatibility type (P>0.2).
Simple linear regression was used to estimate coπelations between postbooster and postchallenge ELISPOT responses, between memory and postchallenge ELISPOT responses, and between logarithmically tiansformed viral loads and ELISPOT frequencies. Comparisons between vaccine and control groups and ^4*0i and non A*01 macaques were performed by means of two-sample t tests with logarithmically transformed viral load and ELISPOT responses. Two-way analyses of variance were used to examine the effects of dose and route of administration on peak DNA/MVA ELISPOTs, on memory DNA/MVA ELISPOTs, and on logarithmically transformed Gag antibody data.
At both peak and memory phases of the vaccine response, the rank order for the height of the ELISPOTs in the vaccine groups was 2.5 mg i.d. > 2.5 mg i.m. > 250 μg i.d. > 250 μg i.m. (Fig. 7B). The IFN-γ ELISPOTs included both CD4 and CD8 cells. Gag- CM9-specific CD8 cells had good lytic activity after restimulation with peptide.
The highly pathogenic SHW-89.6P challenge was administered intrarectally at 7 months after the rMVA booster, when vaccine-raised T cells were in memory (Fig. 7). The challenge stock (5.7 x 109 copies of viral RNA per milliliter) was produced by one intravenous followed by one intrarectal passage in rhesus macaques of the original SHW-89.6P stock (G.B. Karlsson et al. 1997 J Virol 71:4218). Lymphoid cells were harvested from the intrarectally infected animal at peak viremia, CD8-depleted, and mitogen-stimulated for stock production. Before intrarectal challenge, fasted animals were anesthetized (ketamme, 10 mg/kg) and placed on their stomach with the pelvic region slightly elevated. A feeding tube (8Fr (2.7 mm) x 16 inches (41 cm); Sherwood Medical, St. Louis, MO) was inserted into the rectum for a distance of 15 to 20 cm. Following insertion of the feeding tube, a syringe containing 20 intrarectal infectious doses in 2 ml of RPMI- 1640 plus 10% FBS was attached to the tube and the inoculum was slowly injected into the rectum. After delivery of the inoculum, the feeding tube was flushed with 3.0 ml of RPMI without FBS and then slowly withdrawn. Animals were left in place, with pelvic regions slightly elevated, for a period often minutes after the challenge. The challenge infected all ofthe vaccinated and control animals (Fig. 8). However, by 2 weeks after challenge, titers of plasma viral RNA were at least 10-fold lower in the vaccine groups (geometric means of lxlO7 to 5xl07) than in the control animals (geometric mean of 4x108) (Fig. 8 A) (S. Staprans et al. in: Viral Genome Methods K. Adolph, ed. CRC Press, Boca Raton, FL, 1996 pp. 167-184; R. Hofinann-Lehmann et al. 2000 AIDS Res Hum Retroviruses 16:1247).
For the determination of SHW copy number, viral RNA from 150 μl of ACD anticoagulated plasma was directly extracted with the QIAamp Viral RNA kit (Qiagen), eluted in 60 μl of AVE buffer, and frozen at -80°C until SHW RNA quantitation was performed. Five microliters of purified plasma RNA was reverse-transcribed in a final 20- μl volume containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 1 mM each deoxynucleoti.de triphosphate (dNTP), 2.5 μM random hexamers, 20 units MultiScribe RT, and 8 units ribonuclease inhibitor. Reactions were incubated at 25°C for 10 min, followed by incubation at 42°C for 20 min, and inactivation of reverse transcriptase at 99°C for 5 min. The reaction mix was adjusted to a final volume of 50 μl containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 0.4 mM each dNTP, 0.2 μM forward primer, 0.2 μM reverse primer, 0.1 μM probe, and 5 units AmpliTaq Gold DNA polymerase (all reagents from PerkinElmer Applied Biosystems, Foster City, CA). The primer sequences witliin a conserved portion of the SW gag gene are the same as those described previously (S. Staprans et al. in: Viral Genome Methods K. Adolph, ed. CRC Press, Boca Raton, FL, 1996 pp. 167-184). A PerkinElmer Applied Biosystems 7700 Sequence Detection System was used with the PCR profile: 95°C for 10 min, followed by 40 cycles at 93°C for 30 s, and 59.5°C for 1 min. PCR product accumulation was monitored with the 7700 sequence detector and a probe to an internal conserved gag gene sequence: 6FAM- CTGTCTGCGTCATTTGGTGC-Tamra (SEQ ID NO: 8), where FAM and Tamra denote the reporter and quencher dyes. SHW RNA copy number was determined by comparison with an external standard curve consisting of virion-derived SWmac239 RNA quantified by the SW bDNA method (Bayer Diagnostics, Emeryville, CA). All specimens were extracted and amplified in duplicate, with the mean result reported. With a 0.15-ml plasma input, the assay has a sensitivity of 103 RNA copies per milliliter of plasma and a linear dynamic range of 103 to 108 RNA copies (R2 = 0.995). The intraassay coefficient of variation was <20% for samples containing >104 SHW RNA copies per milliliter, and <25% for samples containing 103 to 104 SHW RNA copies per milliliter. To more accurately quantitate low SHW RNA copy number in vaccinated animals at weeks 16 and 20, we made the following modifications to increase the sensitivity of the SHW RNA assay: (i) Virions from <1 ml of plasma were concentrated by centrifugation at 23,000g at 10°C for 150 min before viral RNA extraction, and (ii) a one-step reverse transcriptase PCR method was used (R. Hofmann-Lehmann et al. 2000 AIDS Res Hum Retroviruses 16:1247). These changes provided a reliable quantification limit of 300 SHW RNA copies per milliliter, and gave SHW RNA values that were highly coπelated to those obtained by the first method used (r = 0.91, PO.0001).
By 8 weeks after challenge, both high-dose DNA-primed groups and the low-dose i.d. DNA-primed group had reduced their geometric mean loads to about 1000 copies of viral RNA per milliliter. At this time, the low-dose i.m. DNA-primed group had a geometiic mean of 6x10 copies of viral RNA and the nonvaccinated contiols had a geometric mean of 2 x 106. By 20 weeks after challenge, even the low-dose i.m. group had reduced its geometric mean copies of viral RNA to 1000. Among the 24 vaccinated animals, only one animal, animal number 22 in the low-dose i.m. group, had intermittent viral loads above IxlO4 copies per milliliter (Fig 8D).
By 5 weeks after challenge, all of the nonvaccinated controls had undergone a profound depletion of CD4 cells (Fig 8B). All of the vaccinated animals maintained their CD4 cells, with the exception of animal 22 in the low dose i.m. group (see above), which underwent a slow CD4 decline (Fig. 8E). By 23 weeks after challenge, three of the four control animals had succumbed to AIDS (Fig. 8C). These animals had variable degrees of enterocolitis with diarrhea, cryptosporidiosis, colicystitis, enteric campylobacter infection, splenomegaly, lymphadenopathy, and SW-associated giant cell pneumonia. In contrast, all 24 vaccinated animals maintained their health. Containment of the viral challenge was associated with a burst of antiviral T cells
(Fig. 7 and 9A). At one week after challenge, the frequency of tetramer cells in the peripheral blood had decreased, potentially reflecting the recruitment of specific T cells to the site of infection (Fig. 9A). However, by two weeks after challenge, tetramer"1" cells in the peripheral blood had expanded to frequencies as high as, or higher than, after the rMVA booster (Fig. 7 and 9A). The majority of the tetramer"1" cells produced IFN-γ in response to a 6-hour peptide stimulation (Fig. 9B) (S.L. Waldrop et al. 1997 J Gin Invest 99:1739) and did not have the "stunned" IFN-γ negative phenotype sometimes observed in viral infections (F. Lechner et al. 2000 JExp Med 191:1499). For intracellular cytokine assays, about lxlO6 PBMC were stimulated for 1 hour at 37°C in 5 ml polypropylene tubes with 100 μg of Gag-CM9 peptide (CTPYDTNQM) (SEQ ID NO: 6) per milliliter in a volume of 100 μl RPMI containing 0.1% bovine serum albumin (BSA) and 1 μg of antibody to human CD28 and 1 μg of antibody to human CD49d (Pharmingen, San Diego, CA) per milliliter. Then, 900 μl of RPMI containing 10% FBS and monensin (10 μg/ml) was added, and the cells were cultured for an additional 5 hrs at 37°C at an angle of 5° under 5% CO2. Cells were surface stained with antibodies to CD8 conjugated to PerCP (clone SKI, Becton Dickinson) at 8° to 10°C for 30 min, washed twice with cold PBS containing 2% FBS, and fixed and permeabilized with Cytofix/Cytoperm solution (Pharmingen). Cells were then incubated with antibodies to human CD3 (clone FN-18; Biosource International, Camarillo, CA) and IFN-γ (Clone B27; Phanningen) conjugated to FITC and phycoerythrin, respectively, in Perm wash solution (Pharmingen) for 30 min at 4°C Cells were washed twice with Perm wash, once with plain PBS, and resuspended in 1% paraformaldehyde in PBS. About 150,000 lymphocytes were acquired on the FACScaliber and analyzed with Flo Jo software.
The postchallenge burst of T cells contracted concomitant with the decline of the viral load, By 12 weeks after challenge, virus-specific T cells were present at about one- tenth of their peak height (Figs. 7A and 9A). In contrast to the vigorous secondary response in the vaccinated animals, the naive animals mounted a modest primary response (Figs. 7B and 9A). Tetramer4" cells peaked at less than 1% of total CDS cells (Fig. 9A), and IFN-γ-producing ELISPOTs were present at a mean frequency of about 300 as opposed to the much higher frequencies of 1000 to 6000 in the vaccine groups (Fig. 7B) (P<0.05).
The tetramer4" cells in the control group, like those in the vaccine group, produced IFN-γ after peptide stimulation (Fig. 9B). By 12 weeks after challenge, three of the four controls had undetectable levels of IFN-γ-producing ELISPOTs. This rapid loss of antiviral T cells in the presence of high viral loads may reflect the lack of CD4 help.
T cell proliferative responses demonstrated that virus-specific CD4 cells had survived the challenge and were available to support the antiviral immune response (Fig. 9C). About 0.2 million PBMC were stimulated in triplicate for 5 days with the indicated antigen in 200 μl of RPMI at 37°C under 5% CO2. Supernatants from 293T cells transfected with DNA expressing either SHW-89.6 Gag and Pol or SHW-89.6 Gag, Pol and Env were used directly as antigens (final concentration of ~0.5 μg of p27 Gag per milliliter). Supernatants from mock DNA (vector alone)~transfected cells served as negative controls. On day six, cells were pulsed with 1 μCi of tritiated thymidine per well for 16 to 20 hours. Cells were harvested with an automated cell harvester (TOMTEC, Harvester 96, Model 1010, Hamden, CT) and counted with a Wallac 1450 MICROBETA Scintillation counter (Gaithersburg, MD). Stimulation indices are the counts of tritiated thymidine incorporated in PBMC stimulated with 89.6 antigens divided by the counts of tritiated thymidine incorporated by the same PBMC stimulated with mock antigen.
At 12 weeks after challenge, mean stimulation indices for Gag-Pol-Env or Gag-Pol proteins ranged from 35 to 14 in the vaccine groups but were undetectable in the control group. Consistent with the proliferation assays, intracellular cytokine assays demonstrated the presence of virus-specific CD4 cells in vaccinated but not control animals. The overall rank order ofthe vaccine groups for the magnitude ofthe proliferative response was 2.5 mg i.d. > 2.5 mg i.m. > 250 μg i.d. > 250 μg i.m.
At 12 weeks after challenge, lymph nodes from the vaccinated animals were morphologically intact and responding to the infection, whereas those from the infected controls had been functionally destroyed (Fig. 10). Nodes from vaccinated animals contained large numbers of reactive secondary follicles with expanded germinal centers and discrete dark and light zones (Fig. 10A). By contrast, lymph nodes from the nonvaccinated control animals showed follicular and paracortical depletion (Fig. 10B), while those from unvaccinated and unchallenged animals displayed normal numbers of minimally reactive geπninal centers (Fig. 10C). Germinal centers occupied < 0.05% of total lymph node area in the infected controls, 2% of the lymph node area in the uninfected controls, and up to 18% ofthe lymph node area in the vaccinated groups (Fig. 10D). More vigorous immune reactivity in the low-dose than the high-dose DNA-primed animals was suggested by more extensive germinal centers in the low dose group (Fig. 10D). At 12 weeks after challenge, in situ hybridization for viral RNA revealed rare virus-expressing cells in lymph nodes from 3 of the 24 vaccinated macaques, whereas virus-expressing cells were readily detected in lymph nodes from each o the infected control animals. In the controls, which had undergone a profound depletion in CD4 T cells, the cytomorphology of infected lymph node cells was consistent with a macrophage phenotype.
The prime/boost strategy raised low levels of antibody to Gag and undetectable levels of antibody to Env (Fig. 11). Postchallenge, antibodies to both Env and Gag underwent anamnestic responses with total Gag antibody reaching heights approaching 1 mgJm and total Env antibody reaching heights of up to 100 μg/ml.
Enzyme-linked immimosorbent assays (ELISAs) for total antibody to Gag used bacterially produced SIV gag p27 to coat wells (2 μg per milliliter in bicarbonate buffer).
5 ELISAs for antibody to Env antibody used 89.6 Env produced in transiently transfected 293T cells and captured with sheep antibody against Env (catalog number 6205; International Enzymes, Fairbrook CA). Standard curves for Gag and Env ELISAs were produced with serum from a SHW-89.6-infected macaque with known amounts of immunoglobulin G (IgG) specific for Gag or Env. Bound antibody was detected with
10 peroxidase-conjugated goat antibody to macaque IgG (catalog # YNGMOIGGFCP; Accurate Chemical, Westbury, NY) and TMB substrate (Catalog # T3405; Sigma, St. Louis, MO). Sera were assayed at threefold dilutions in duplicate wells. Dilutions of test sera were performed in whey buffer (4% whey and 0.1% tween 20 in IX PBS). Blocking buffer consisted of whey buffer plus 0.5% nonfat dry milk. Reactions were stopped with
'5 2M H2SO4 and the optical density read at 450 am. Standard curves were fitted and sample concentrations were interpolated as μg of antibody per ml of serum using SOFTmax 2.3 software (Molecular Devices, Sunnyvale, CA),
By 2 weeks after challenge, neutralizing antibodies for the 89.6 immunogen, but not the SHW-89.6P challenge, were present in the high-dose DNA-primed groups (geometric
.0 mean titers of 352 in the i.d. and 303 in the i.m. groups) (Fig. 11C) (D.C. Montefϊori et αl. 1988 J Gin Microbiol 26:231). By 5 weeks after challenge, neutralizing antibody to 89.6P had been generated (geometric mean titers of 200 in the high-dose i.d. and 126 in the high- dose i.m. group) (Fig. 11D) and neutralizing antibody to 89.6 had started to decline. By 16 to 20 weeks after challenge, antibodies to Gag and Env had fallen in most animals.
5 Our results demonstrate that a multiprotein DNA MVA vaccine can raise a memory immune response capable of controlling a highly virulent mucosal immunodeficiency virus challenge. Our levels of viral control were more favorable than have been achieved using only DNA (M.A. Egan et αl. 2000 J Virol 74:7485) or rMVA vaccines (I. Ourmanov et αl. 2000 J Virol 74:2740) and were comparable to those obtained for DNA immunizations
0 adjuvanted with interleukin-2 (D.H. Barouch et αl 2000 Science 290:486). All of these previous studies have used more than three vaccine inoculations, none have used mucosal challenges, and most have challenged at peak effector responses and not allowed a prolonged post vaccination period to test for "long term" efficacy. The dose of DNA had statistically significant effects on both cellular and humoral responses (PO.05), whereas the route of DNA administration affected only humoral responses, hitradermal DNA delivery was about 10 times more effective than i.m. inoculations for generating antibody to Gag (P = 0.02). Neither route nor dose of DNA appeared to have a significant effect on protection. At 20 weeks after challenge, the high- dose DNA-primed animals had slightly lower geometric mean levels of viral RNA (7xl02 and 5xl02) than the low-dose DNA-primed animals (9xl02 and lxlO3).
The DNA/MVA vaccine controlled the infection, rapidly reducing viral loads to near or below 1000 copies of viral RNA per milliliter of blood. Containment, rather than prevention of infection, affords the opportunity to establish a chronic infection (H.L. Robinson et al. 1999 Nat Med 5:526). By rapidly reducing viral loads, a multiprotein DNA/MVA vaccine will extend the prospect for long-term non-progression and limit HW transmission (J.W. Mellors et al. 1996 Science 272:1167; T.C. Quinn et al. 2000 NEngl J Med 342:921). EXAMPLE 2
MVA Expressing Modified HIV Env, Gag, and Pol Genes This disclosure describes the construction of a modified vaccinia Ankara (MVA) recombinant virus, MV A/HIV clade B recombinant virus expressing the HW strain ADA env and the HXB2 gag pol (MVA/HW ADA env + HXB2 gag pol). For amplification, the lab name of MVAJHTV 48 will be used, which denotes the plasmid from which the construct comes.
The HW gag-pol genes were derived from the Clade B infectious HXB2 virus. The gag-pol gene was truncated so that most of the integrase coding sequences were removed and amino acids 185, 266, and 478 were mutated to inactivate reverse transcriptase, inhibit strand transfer activity, and inliibit the RNaseH activity, respectively. The Clade B CCR5 tropic envelope gene was derived from the primary ADA isolate; TTTTTNT (SEQ ID NO: 14) sequences were mutated without changing coding capacity to prevent premature transcription termination and the cytoplasmic tail was truncated in order to improve surface expression, immunogenicity, and stability of the MVA vector. The HW genes were inserted into a plasmid transfer vector so that gag-pol gene was regulated by the modified H5 early/late vaccinia virus promoter and the env gene was regulated by the newly designed early/late Psyn II promoter to provide similar high levels of expression. A self- deleting GUS reporter gene was included to allow detection and isolation of the recombinant virus. The HW genes were flanked by MVA sequences to allow homologous recombination into the deletion 3 site so that the recombinant MVA would remain TK positive for stability and high expression in resting cells. The recombinant MVA was isolated and shown to express abundant amounts of gag-pol-env and to process gag. Production of HW-like particles was demonstrated by centrifugation and by electron microscopy. The presence of env in the HW-like particles was demonstrated by immunoelectron microscopy.
Table of Sequences
Plasmid Transfer Vector
The plasmid transfer vector used to make the MVA recombinant virus, pLW-48, (Figure 15) by homologous recombination was constructed as follows:
1. From the commercially obtained plasmid, pGem-4Z (Promega), flanking areas on either side of deletion III, designated flank 1 and flank 2, containing 926 and 520 base pairs respectively, were amplified by PCR from the MVA stains of vaccinia virus. Within these flanks, a promoter, the mH5, which had been modified from the originally published sequence by changing two bases that had been shown by previously published work to increase the expression ofthe cloned gene, was added.
2. A clade B gag pol (Figure 16) was truncated so that the integrase was removed and was cloned into the plasmid so that it was controlled by the mH5 promoter. This gene contained the complete HXB2 sequence o the gag. The pol gene has reverse transcriptase safety mutations in amino acid 185 within the active site of RT, in amino acid 266 which inhibits strand transfer activity, and at amino acid 478 which inhibits the RNaseH activity, hi addition, the integrase gene was deleted past EcoRI site. 3. A direct repeat of 280 basepairs, corresponding to the last 280 base pairs of MVA flank 1, was added after flank 1. 4. The pll promoter and GUS reporter gene were added between the two direct repeats of flank 1 so that this screening marker could initially be used for obtaining the recombinant virus, yet deleted out in the final recombinant virus (Scheiflinger, F. et al. 1998 Arch Virol 143:467-474; Carroll, M.W. and B. Moss 1995 BioTechniques 19:352- 355).
5. A new promoter, Psyn II, was designed to allow for increased expression of the ADA env. The sequence of this new early/late promoter is given in Figure 17.
6. A truncated version of the ADA envelope with a silent 5TNT mutation was obtained by PCR and inserted in the plasmid under the control of the Psyn II promoter. The envelope was truncated in the cytoplasmic tail of the gp41 gene, deleting 115 amino acids of the cytoplasmic tail. This truncation was shown to increase the amount of envelope protein on the surface of infected cells and enhance immunogenicity of the envelope protein in mice, and stability ofthe recombinant virus in tissue culture. Recombinant MVA Construction 1. MVA virus, which may be obtained from ATCC Number VR-1508, was plaque purified three times by terminal dilutions in chicken embryo fibroblasts (CEF), which were made from 9 day old SPF Premium SPAFAS fertile chicken eggs, distributed by B and E Eggs, Stevens, PA.
2. Secondary CEF cells were infected at an MOI of 0.05 of MVA and transfected with 2 μg of pLW-48, the plasmid described above. Following a two-day incubation at
37°C, the virus was harvested, frozen and thawed 3x, and plated out on CEF plates.
3. At 4 days, those foci of infection that stained blue after addition of X-gluc substrate, indicating that recombination had occurred between the plasmid and the infecting virus, were picked and inoculated on CEF plates. Again, those foci that stained blue were picked.
4. These GUS containing foci were plated out in triplicate and analyzed for GUS staining (which we wanted to now delete) and ADA envelope expression. Individual foci were picked from the 3rd replicate plates of those samples that had about equal numbers of mixed populations of GUS staining and nonstaining foci as well as mostly envelope staining foci.
5. These foci were again plated out in triplicate, and analyzed the same way. After 5 passages, a virus was derived which expressed the envelope protein but which had deleted the GUS gene because of the double repeat. By immunostaining, this virus also expressed the gag pol protein.
Characterization of MVA Recombinant Vims, MVA/HW 48
1. Aliquots of MVA/HW 48 infected cell lysates were analyzed by 5 radioimmunoprecipitation and immunostaining with monoclonal antibodies for expression of both the envelope and gag pol protein, hi both of these tests, each of these proteins was detected.
2. The recombinant virus was shown to produce gag particles in the supernatant of infected cells by pelleting the 3sS-labeled particles on a 20% sucrose cushion.
0 3. Gag particles were also visualized both outside and budding from cells as well as within vacuoles of cells in the electron microscope in thin sections. These gag particles had envelope protein on their surface.
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer, and all amino
5 acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to
!0 at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well lαiown in the art. As is also lαiown in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that
!5 the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. Summary
In summary, we have made a recombinant MVA virus, MVA HW 48, which has
JO high expression of the ADA truncated envelope and the HXB2 gag pol. The MVA recombinant virus is made using a transiently expressed GUS marker that is deleted in the final virus. High expression of the ADA envelope is possible because of a new hybrid early/late promoter, Psyn II. In addition, the envelope has been truncated because we have shown truncation of the envelope enhances the amount of protein on the surface of the infected cells, and hence enhances immunogenicity; stability of the recombinant is also enhanced. The MVA recombinant makes gag particles which have been shown by pelleting the particles through sucrose and analyzing by PAGE. Gag particles with envelope protein on the surface have also been visualized in the electron microscope.
EXAMPLE 3 Additional Modified or Synthetic Promoters Designed for Gene Expression in MVA
Or Other Poxviruses
Additional modified or synthetic promoters were designed for gene expression in MVA or other poxviruses. Promoters were modified to allow expression at early and late times after infection and to reduce possibility of homologous recombination between identical sequences when multiple promoters are used in same MVA vector. Promoters are placed upstream of protein coding sequence. m7.5 promoter (SEQ ID NO: 10): CGCTTTTT ATAGTAAGTTTTTC ACCC ATAAATAATAAAT ACAAT AATT AATTTCT CGTAAAAATTGAAAAACTATTCTAATTTATTGCACGGT
Psyn II promoter (SEQ ID NO: 2): TAAAAAATGAAAAAATATTCTAATTTATAGGACGGTTTTGATTTTCTTTTTTTCT
ATGCTATAAATAATAAATA Psyn III promoter (SEQ ID NO: 11):
TAAAAATTGAAAAAATATTCTAATTTATAGGACGGTTTTGATTTTCTTTTTTTCT
ATACTATAAATAATAAATA
Psyn IV promoter (SEQ ID NO: 12):
TAAAAATTGAAAAACTATTCTAATTTATAGGACGGTTTTGATTTTCTTTTTTTCT ATACTATAAATAATAAATA
Psyn V promoter (SEQ ID NO: 13):
AAAAAATGATAAAGTAGGTTCAGTTTTATTGCTGGTTTAAAATCACGCTTTCGA GTAAAAACTACGAATATAAAT
EXAMPLE 4 Tables A-F
Table A: MVA/48 immunization - guinea pigs. Groups of guinea pigs were immunized at days 0 and 30 with 1 xlO8 infectious units of MVA/48 by either the intramuscular (EVf) or intradermal (ID) route. As a control another group was immunized TM with the same dose of non-recombinant MVA. Sera taken before as well as after each immunization were analyzed for neutralizing activity against HW-l-MN. Titers are the reciprocal serum dilution at which 50% of MT-2 cells were protected from virus-induced killing. Significant neutralizing activity was observed in all animals after the second immunization with MVA/48 (day 49).
Table B: Frequencies of HW-l gag-specific T cells following immunization of mice with MVA/48.
Groups of BalbC mice were immunized at days 0 and 21 with 1 xlO7 infectious units of MVA 48 by one of three routes: intraperitoneal (IP), intradermal (ID), or intramuscular (TM). A control group was immunized with non-recombinant MVA. At 5 weeks after the last immunization, splenocytes were prepared and stimulated in vitro with an immunodominant peptide from HW-l p24 for 7 days. The cells were then mixed either with peptide-pulsed P815 cells or with soluble peptide. Gamma interferon-producing cells were enumerated in an ELISPOT assay. A value of >500 was assigned to wells containing too many spots to count. Strong T cell responses have been reported in mice immunized IP with other viruses. In this experiment, IP immunization of mice with MVA/48 elicited very strong HW-l gag-specific T cell responses.
Table C: DNA prime and MVA/48 boost - total ELISPOTS per animal.
Ten rhesus macaques were primed (weeks 0 and 8) with a DNA vaccine expressing HW-l antigens including Ada envelope and HXB2 gagpol. At week 24 the animals were boosted intramuscularly with 1 xlO8 infectious units of MVA/48. Fresh peripheral blood mononuclear cells (PBMC) were analyzed for production of gamma interferon in an ELISPOT assay as follows: PBMC were incubated for 30-36 hours in the presence of pools of overlapping peptides corresponding to the individual HW-l antigens in the vaccines. The total number of gamma interferon-producing cells from each animal is shown in the table. T cell responses to DNA vaccination were limited (weeks 2-20). However, boosting with MVA 48 resulted in very strong HW-l -specific T cell responses in all animals (week 25).
Table D: Antibody response following immunization of macaques with MVA/SHW KB9. Groups of rhesus macaques were immunized with 2 xlO8 infectious units of MVA SHW-KB9 at weeks 0 and 4 by one of several routes: Tonsilar, intradermal (ID), or intramuscular (IM). Another group was immunized with non-recombinant MVA using the same routes. Serum samples from 2 weeks after the second immunization were analyzed for binding to KB9 envelope protein by ELISA and for neutralization of SHW-89.6P and SHW-89.6. In the ELISA assay, soluble KB9 envelope protein was captured in 96 well plates using an antibody to the C-terminus of gpl20. Serial dilutions of sera were analyzed and used to determine the endpoint titers. Neutralization of SHW-89.6P and SHW-89.6 was determined in an MT-2 cell assay. Titers are the reciprocal serum dilution at which 50%) of the cells were protected from virus-induced killing. In in vitro neutralization assays, SHW-89.6P and SHW-89.6 are heterologous, i.e. sera from animals infected with one of the viruses do not neutralize the other virus. Thus, two immunizations with MVA/SHW-KB9 elicited good ELISA binding antibodies in all animals and neutralizing antibodies to the homologous virus (SHW-89.6P) in some animals. In addition, heterologous neutralizing antibodies were observed in a subset of animals.
Table E: Frequencies of gag CM-9-specific CD3/CD8 T cells following immunization of macaques with MVA/SHW-KB9.
Groups of MamuA*01 positive rhesus macaques were immunized with 2 xlO infectious units of MVA/SHW-KB9 at weeks 0 and 4 by one of several routes: tonsilar, intradermal (ID), or intramuscular IM). Another group was immunized with non- recombinant MVA. The frequencies of CD3+/CD8+ T cells that bound tetrameric complex containing the SW gag-specific peptide CM9 were determined by flow cytometry at various times after each immunization. Time intervals were as follows: la, lb, and Id were one, two, and four weeks after the first immunization, respectively; 2a, 2b, 2c, and 2d were one, two, three, and twelve weeks after the second immunization, respectively. Values above background are shown in bold face. Strong S TV gag-specific responses were observed after a single immunization with MVA/SHW-KB9 in all immunized animals. Boosting was observed in most animals following the second immunization. In addition, measurable tetramer binding was still found twelve weeks after the second immunization. Table F: Frequencies of specific T cells following immunization of macaques with
MVA/SHW KB9.
Groups of macaques were immunized with MVA/SHW-KB9 as described above. MVA/SHW-KB9 expresses 5 genes from the chimeric virus, SHW-89.6P: envelope, gag, polymerase, tat, and nef. Thus, the frequencies of T cells specific for each ofthe 5 antigens were analyzed using pools of peptides coπesponding to each individual protein. Fresh PBMC were stimulated with pools of peptides for 30-36 hours in vitro. Gamma interferon- producing cells were enumerated in an ELISPOT assay. The total number of cells specific for each antigen is given as "total # spots", hi addition, the number of responding animals and average # of spots per group is shown. PBMC were analyzed at one week after the first immunization (la) and one week after the second immunization (2a). Another group of 7 animals was immunized with non-recombinant MVA. In these animals, no spots above background levels were detected. Thus, a single immunization with MVA/SHW- KB9 elicited strong SHW-specific T cell responses in all animals. Gag and envelope responses were the strongest; most animals had responses to gag, all animals had responses to envelope. The Elispot responses were also observed after the second immunization with MVA/SHW-KB9, albeit at lower levels. At both times, the rank order of responses was: tonsilar > ID > BVI. We show good immune response to nef and some immune response to
I3.Ϊ.
TABLE A. MVA/48 immunisation - guinea pigs HIV-MN neutralising antibody - reciprocal titer
TABLE B. Frequencies of HIV-gag-specific T cells following immunization of mice with MVA/48
TABLE C. DNA prime and MVA/48 boost. Total ELISPOTS per Animal
DNA primes were at 0 and 8 weeks and MVA/48 boost was at 24 weeks
1 < = Background (2x the number of ELISPOTs in the unstimulated control + 10)
2 Costimulatory antibodies were added to the ELISPOT incubations * Animals from this bleed date exhibited higher than usual ELISPOTs.
TABLE D. Antibody response following immunization of macaques with MVA SHIV
KB9
TABLE E. Frequencies of gag CM9-specific CD3/CD8 T cells following immunization of macaques with MVA/SHIV KB9
TABLE F. Frequencies of specific T cells following immunization of macaques with MVA/SHIV KB9
EXAMPLE 5
Construction and Pre-clinical Immunogenicity of a Recombinant MVA Vaccine
Expressing Subtype D HIV-1 Env, Gag and Pol for Use in Uganda.
Recombinant MVA vaccines have been successful in generating SW and SHW specific humoral and CDS T cell responses in non-human primates and, alone or in combination with DNA vaccines, have provided protection in rhesus macaques from disease after pathogenic SHW challenge. An overall program goal is to conduct clinical vaccine trials in Africa using vaccines that induce both neutralizing antibody and CD8 T cell specific responses and that are based upon representative full-length HW-l sequences isolated from the target vaccine cohorts. The predominant incident and prevalent HW-l subtype in Uganda is subtype D. Several R5 subtype D HW-l strains were selected and used to prepare recombinant MVA vaccines expressing env, gag, protease and RT. Initially, multiple env and gag/pol clones from 3 pure Ugandan subtype D isolates were selected. These sequences were separately cloned into pCR2.1 and tested for expression levels in-vitro by immunoprecipitation, and for envelope function as assessed by envelope- mediated fusion with CD4 and CCR5 or CXCR4 expressing cells. Based on these in-vitro analyses, several R5 subtype D env and gag/pol sequences were selected and cloned into MVA shuttle plasmids containing GFP and the modified H5 promoter for sequential cloning into deletions II and III, respectively, of MVA. The parent MVA used was a 1974 stock chosen to eliminate FDA concerns regarding potential BSE contamination. Several recombinant MVA (rMVA-UGD) expressing subtype D env and gag/pol were prepared in primary CEF cells using gamma-irradiated FBS from a USD A approved source and selected using GFP expression. These rMVA-UGD were further plaque-purified and amplified to titers sufficient for in-vivo immunogenicity studies. Pre-clinical humoral and cellular immunogenicity ofthe various rMVA-UGD were then assessed in BALB/c mice. MVA Expressing Altered HIV-1 Envelope, Gag, and Polymerase Genes from
Ugandan Clade D This example describes the construction of 5 recombinant Modified Vaccinia Virus Ankara (MVA) viruses expressing envelope (env) and gagpol genes from HW-l clade D isolates from Uganda. Sequences from Ugandan Clade D:
Env and gagpol genes from three Ugandan clade D HW-l isolates were used:
Env and gagpol genes were PCR amplified from Ugandan HW-l clade D isolates by short term co-cultures on normal human PBMC (Harris et al. 2002 AIDS Research and Human Retroviruses 18:1281) using the oligonucleotides shown in Table G and cloned into pCR2.1-TOPO (Invitrogen). (HW-I infected individuals contain a population or quasi-species of related but distinct viruses. Upon co-culture, multiple viruses can emerge such that the sequences of individual amplified genes from the co-culture may differ from the sequence of the full genome.) The resulting amplified env genes have a C-terminal deletion of 115 amino acids that was previously shown to enhance expression and yield a more stable recombinant virus. The resulting gagpol genes have a deletion of the entire integrase and Rnase H portions of the genes. Within both the env and gagpol genes, several mutations were made by site-directed mutagenesis (Quik Change from Stratagene). In the env genes, silent mutations were made to eliminate two naturally occurring vaccinia virus early transcription termination signals (TTTTTNT, SEQ ID NO: 14) (Earl et. al 1990 J. Virol 64:2451). hi the pol genes, two mutations were made in separate locations in the active site of reverse transcriptase to abolish enzymatic activity (Larder et. al 1987 Nature 327:716)(see Tables H(i) & (ii) for details on changes made to env and gagpol genes). PCR2.1-TOPO plasmids containing the amplified genes were first characterized with respect to the orientation ofthe gene. Clones in which the gene was oriented properly with respect to the T7 promoter were chosen and protein expression was analyzed as previously described (Earl et al. 1997 J Virol 71:2674). Briefly, BS-C-1 cells were infected with vTF7-3 (Fuerst et al 1986 PNAS USA 83:8122), a recombinant vaccinia virus expressing T7 RNA polymerase, transfected with a plasmid, and metabohcally labeled. Cell lysates were subjected to immunoprecipitation with serum pooled from several HW-l clade D-infected individuals. Proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. One env and one gagpol DNA clone from each clade D isolate was chosen for construction of recombinant MVA viruses. DNA sequencing was performed to confirm the integrity of each gene. Sequences are presented in Appendix 1.
Cloning into shuttle plasmids:
Two MVA shuttle plasmids, pLAS-T and pLAS-2 (Figure 18), were used for construction of recombinant MVA viruses. DNA sequences of both plasmids are presented in Appendix 2. In both plasmids, the foreign gene is driven by the modified H5 promoter. In addition, both plasmids contain a cassette with the gene for green fluorescent protein (GFP) driven by the vaccinia pi 1 promoter. This cassette is flanked by direct repeats that will readily recombine to eliminate GFP during virus propagation. Thus, GFP is used as a positive screening marker in early rounds of plaque purification, and as a negative screening marker in final recombinant virus selection (Figure 22). MVA flanking sequences in pLAS-1 and pLAS-2 direct recombination into deletion III (Del III) and deletion II (Del II) of MVA, respectively.
Gagpol genes from 2 isolates (99UGA03349 and 99UGA07412) were cloned separately into pLAS-1 for insertion into Del III of MVA. The plasmids were named pLAS-1/UGD/Bgag and pLAS-1/UGD/Cgag (Figure 19 & Table I). When the env gene is cloned into the Notl restriction site, a short open reading frame precedes the env open reading frame. This open reading frame is out of frame with env and terminates at approximately nucleotide 75 in the env gene. Env genes from three isolates (99UGA03349, 99UGA07412, 98UG57128) were cloned separately into MVA shuttle plasmid pLAS-2, for insertion into Del II of MVA. Plasmids were named pLAS-2/UGD/Benv, pLAS-2/UGD/Cenv, and pLAS-2/UGD/Denv (Figure 20 & Table I). When the env gene is cloned into the Notl restriction site, a short open reading frame precedes the env open reading frame. This open reading frame is out of frame with env and terminates at approximately nucleotide 75 in the env gene.
Foreign genes in pLAS-1 and pLAS-2 recombine into the vaccinia genome in the same orientation as the suπounding vaccinia genes. To test the effect of reversing the orientation of the env gene on the level of gene expression and stability of viruses, two of the env genes and their promoters were excised from pLAS-2 with restriction endonucleases BspEl and EcoRV; sticky ends were filled in with Klenow enzyme; and the fragments were then reinserted into pLAS-2 the opposite orientation (Figure 21). Plasmids were named pLAS-2/UGD/revCenv and pLAS-2/UGD/revDenv (Table I). Recombinant MVA construction:
Parent MVA: MVA 1974/NIH Clone 1 was used as the parent for all recombinant viruses. It was derived from a stock of MVA at passage 572, prepared on 2/22/1974 in the laboratory of A.Mayr in Germany. After receipt in the Laboratory of Viral Diseases, this stock was passaged a total of 6 times in chicken embryo fibroblast (CEF) cells, including 3 clonal purifications. Amplification was performed on the final, clonally purified virus. All CEF cells were derived from specific pathogen-free (SPAFAS) eggs.
Recombinant viruses expressing gagpol: CEF cells were infected with MVA 1974/NIH Clone 1 and transfected with either pLAS-1/UGD/Bgag or pLAS-1/UGD/Cgag for insertion into Del HI. Two to three rounds of plaque purification were performed based on GFP expression. Further rounds of plaque purification were performed by picking plaques based on lack of GFP expression and concomitant positive gag expression as measured by immunostaining using a monoclonal antibody to HW-l p24 (183-H12-5C; obtained from the NIH AIDS Research and Reference Reagent Program) (Figure 22). Recombinant gagpol-expressing viruses were amplified and characterized for gag expression by immunoprecipitation as described above. The two viruses were named MVA/UGD/Bgag and MVA/UGD/Cgag. These viruses were then used as the parent in making gagpol/env recombinant viruses (see below).
Recombinant viruses expressing gagpol and env: Recombinant viruses, MVA/UGD/Bgag and MVA/UGD/Cgag were used as parent viruses for insertion of e/?v genes. Thus, CEF cells were infected with either MVA/UGD/Bgag or MVA/UGD/Cgag and subsequently transfected with one of the pLAS-2-env-containing plasmids described above (Figure 23 & Table I). As above, the first two rounds of plaque purification were performed based on GFP expression, hi subsequent rounds of purification, plaques were selected based on loss of GFP expression and positive gag and env expression as measured by immunostaining in duplicate cultures (Figure 22). A total of 5 gagpol/env-expressing viruses (MVA/UGD-1 through -5) were amplified and characterized (Table J). Characterization of recombinant MVA/UGD viruses: The 5 MVA/UGD viruses have been characterized for gene expression and function. Immunoprecipitation of env and gag proteins is shown in Figure 24. BS-C-1 cells were infected with individual recombinant viruses at a multiplicity of infection of 10, metabohcally labeled, and lysates were subjected to immunoprecipitation with a pool of sera from HW-l clade D infected individuals. Viruses expressing gagpol only (MVA UGD/Bgag and Cgag) were included, as was non-recombinant MVA as a negative control and MVA/CMDR as a positive control. The latter virus expresses gagpol/env from a Clade E HW-l isolate. All viruses produced high levels of gag protein and efficient processing into p24 was observed, hi addition, all env-expressing viruses produced high levels of env protein (gp 160).
Figure 25 demonstrates that the gag and env proteins produced by the MVA/UGD viruses are functional. Virus-like particles were obtained by centrifugation of the supernatant of infected cells through a sucrose cushion (Karacostas et al. 1993 Virology 193:661). Pelleted material was then separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography (Panel A). As seen, p55 and p24 gag proteins were found in the pellet indicating that virus-like particles were formed. Panel B shows results of an assay in which env-expressing cells (infected with MVA/UGD virus) were mixed with cells expressing CD4 and co-receptor (X4 or R5) (Nussbaum, Broder, & Berger 1994 J. Virol 68:5411). Fusion was measured by beta-galactosidase activity in cell lysates. As shown, all five MVA/UGD viruses induced fusion with CD4/R5-expressing cells.
Immunogenicity of recombinant MVA UGD viruses (study 1): Groups of Balb/c mice were immunized with individual MVA/UGD viruses, non- recombinant MVA (negative control), or MVA/CMDR (positive control - expressing clade E gagpol/env) at weeks 0 and 3. The dose was 107 infectious units per immunization and the route was intraperitoneal. Humoral and cell mediated responses were measured and are shown in Figures 26-28.
Antibody responses after 2 immunizations are shown in Figure 26. Reciprocal endpoint ELISA titers to p24 at various times after immunization are shown in Panel A. All UGD viruses elicited gag-specific antibodies after 2 immunizations. Env-specific responses are shown in Panel B. hi this experiment, pooled sera from groups of mice were used to immunoprecipitate metabohcally labeled, autologous gpl 60 proteins. As seen, sera from mice immunized with MVA/UGD- 1, -3, and -4 reacted with gpl 60 (the other viruses were not tested in this assay). Reciprocal endpoint titers to gpl 40 env at various times after immunization are shown in Panel C All UGD viruses elicited env-specific antibodies after 2 immunizations.
T cell responses were measured with several assays. First, gag and pol peptide- specific intracellular interferon gamma (IFN- γ) responses were measured by intracellular cytokine staining. Splenocytes were collected 3 weeks after immunization, stimulated in- vitro for 7 days, and then cultured overnight with peptide-pulsed P815 cells. Brefeldin A was added to prevent secretion of INF- γ. CD3 positive, CD8 positive, IFN-γ positive cells were enumerated by flow cytometry. Analyses were performed after one and two immunizations. Both gag- and pol-specific responses were observed after two immunizations (Figure 27) (samples from animals immunized with MVA/UGD-2, and 5 were not assayed). Second, gag- and pol-specific INF- γ responses were measured by ELISPOT (Figure 28 A & B). Briefly, splenocytes from immunized mice were mixed with gag or pol peptide-pulsed P815 cells in 96-well nitrocellulose plates coated with anti-IFN- γ antibody. After overnight incubation, spots were visualized by sequential incubation with anti-IFN- γ biotin antibody, stiaptavidin-HRP, and AEC substrate. Spots were enumerated using a Zeiss ELISPOT reader. Gag peptide-specific responses were found after one immunization and were boosted in most groups after the second immunization. Pol peptide-specific responses were found in several groups after two immunizations. Third, gag peptide-specific responses were measured by tetramer staining (H-2Kd gag LAI tetiamer: AMQMLKETI, SEQ ID NO: 63) (Figure 29). Splenocytes were stimulated in vitro with either gag peptide or MVA/CM240gagpol, a recombinant virus expressing a clade E gagpol. CD3 positive, CD8 positive, tetramer positive cells were enumerated by flow cytometry. Positive tetramer staining was observed with cells from several groups of mice. Immunogenieity of recombinant MVA UGD viruses (study 2):
A second mouse immunogenicity study was performed to confirm the humoral and cellular immunogenicity of MVA/UGD-3 and MVA/UGD-4. BALB/c mice (10 per group) were administered intraperitoneal immunizations of 107 infectious units of MVA at weeks 0 and 3. Five mice per group were sacrificed two weeks after the 1st and 2nd immunizations and spleens were removed for assessment of cellular immunogenicity. Sera were collected from each mouse at weeks -1, 0, 1, 2, 3, 4 and 5. Splenocytes and sera were pooled together by group. HW gag-specific serum IgG responses were detected from all MVA/UGD-irnmunized groups after the 2nd immunization (Figure 30). These gag-specific responses were predominantly of subclass IgG2a for both MVA/UGD-3 and MVA/UGD-4 demonstrating a Thl -type response (Table Q).
HW-specific cell-mediated immunity was assessed by four separate assays: (1) intracellular IFN-γ staining by flow cytometry (ICS), (2) IFN- γ secretion by ELISPOT, (3) gag-peptide specific tetramer staining and (4) cytotoxic T lymphocyte (CTL) killing. (1) ICS: Splenocytes were collected two weeks after the 1st immunization, stimulated for 7 days with MVA-infected P815 cells and then incubated overnight with P815 cells pulsed with a gag or pol peptide previously shown to be target of CD8 T cells in BALB/c mice (Casimiro et al. 2002 J Virol 76:185). Brefeldin A was included in the overnight incubation to prevent cytokine secretion. Both HW gag- and pol-specific responses were detected for the MVA/UGD-immunized, but not control immunized, mice as evidenced by the production of intracellular INF- γ after peptide stimulation (Figure 31 A). For example, 8.5% and 5% of splenocyte lymphocytes from MVA/UGD-4-immunized mice were positive for gag and pol, respectively. Similar results were obtained for the MVA/UGD immunized mice after the 2nd immunization (Figure 31B). (2) IFN- γ ELISPOT: HW gag- specific IFN- γ responses were detected by ELISPOT without prior in-vitro stimulation after both the 1st (Figure 32A) and 2nd immunization (Figure 32B) with a boost detected after the 2nd immunization. HW gag-specific responses were stronger than the pol-specific responses. HW pol-specific responses were detectable after a 7-day in-vitro stimulation with P815 cells pulsed with pol peptide (Figure 32C). (3) Tetramer staining: HIV gag peptide-specific responses were measured by tetramer staining (H-2Kd gag LAI tetramer: AMQMLKETI, SEQ ID NO: 63) (Figure 33). Splenocytes were stained pre or post a 7- day in-vitro stimulation with P815 cells either pulsed with gag peptide or infected with MVA/CM240gag/pol, a recombinant virus expressing a subtype E gagpol. CD3 positive/CD8 positive/tetramer positive cells were enumerated by flow cytometry. Positive tetramer staining was observed for all of the MVA/UGD immunized groups both pre-WS and post-WS with MVA-infected P815 cells. (4) CTL: Splenocytes removed 2 weeks after the 1st immunization were stimulated in-vitro with MVA/CME (a recombinant MVA expressing env and gagpol from a subtype E HW-l isolate) infected P815 cells for 7 days and tested for the ability to lyse P815 cells pulsed with gag peptide. Splenocytes from all MVA/UGD, but not MVA control, immunized mice efficiently lysed peptide-pulsed P815 cells at E:T ratios of 20:1 (Figure 34).
EXAMPLE 6 Construction and Pre-clinical Immunogenicity of a Recombinant MVA Vaccine Expressing Subtype A HIV-1 Env, Gag and Pol for Use in Kenya.
As part ofthe overall program goal to conduct clinical vaccine trials in Africa, HW- 1 sequences from Kenya were selected. The predominant incident and prevalent HW-l subtype in Kenya is subtype A. Several R5 subtype A HW-l strains were selected and used to prepare recombinant MVA vaccines expressing env and gagpol (gag, protease and RT). One gagpol and two env clones from pure Kenyan subtype A isolates were selected.
All the steps described in EXAMPLE 5 for construction of subtype D recombinant MVA viruses were followed for the selected subtype A clones. These include: PCR amplification of truncated genes and cloning into pCR2.1.
Testing for in vitro expression.
Testing for env and gag function.
Site directed mutagenesis in env to eliminate vaccinia virus early transcription termination sites. Site directed mutagenesis in pol to inactivate enzymatic activity.
Cloning into MVA shuttle plasmids pLAS-1 (gagpol) and pLAS-2 (env).
Recombination of gagpol into MVA 1974/NIH Clone 1 using primary CEF cells.
Recombination of env into the recombinant virus expressing gag to produce a single virus expressing both gagpol and env. Recombination of env into MVA 1974/NIH Clone 1 to produce a virus expressing env only.
In addition to the mutations described in EXAMPLE 5 and utilized with the Ugandan subtype D env genes, two other mutations were introduced into one of the Kenyan clade A env genes (KNH1144). First, the tyr at position 717 was mutated using site directed mutagenesis to either ala or ser. This mutation has been shown to increase cell surface expression of env proteins (Rowell et .al. 1995 J Immunol 155:473; LaBranche et al. 1995 J Virol 69:5217). Second, the env protein was further truncated at the C-terminus just prior to the transmembrane domain yielding a soluble, secreted form of the protein. Published studies have shown that immunization with this fonn ofthe env protein results in enhanced antibody production as compared to membrane bound env.
The specifics of deletions and mutations in env and gagpol genes for KEA isolates are given in Table K. Plasmids and viruses expressing KEA env and gagpol genes are given in Table L and M. _ 'i...
Characterization of recombinant MVA KEA viruses:
The MVA/KEA viruses were characterized for gene expression and function, hmnunoprecipitation of env and gag protiens is shown in Figure 35. BSC-1 cells were infected with individual recombinant viruses at a multiplicity of infection of 10,
5 metabohcally labeled, and lysates were subjected to imunoprecipitation with a pool of antibodies including: monoclonal antibody T24 (env), monoclonal antibody 183-H12-5C (gag), and pooled HW-1+ sera. MVA/UGD and WR/vEJW-1 were included as positive controls for env and gag expression, respectively. All MVA/KEA viruses express high levels of env and/or gag, as expected.
10 Virus-like particles were obtained by centrifugation of the supernatant of infected cells through a sucrose cushion (Karacostas et al. 1993 Virology 193:661), Gag p24 protein was found in the pelleted material indicating the formation of virus-like particles (Figure 36).
A cell-cell fusion assay was used to assess the function of expressed, membrane
15 bound env. In this assay env-expressing cells (infected with MVA/KEA virus) were mixed with cells expressing CD4 and co-receptor (X4 or R5) (Nussbaum, et al. 1994 J Virol 68:5411). Fusion was measured by beta-galactosidase activity in cell lysates. All viruses expressing membrane bound env induced fusion with CD4/R5-expressing cells (Figure 37).
20 Immunogenicity of env and gag in mice:
The 5 recombinant MVA viruses expressing env, gag and pol from Kenyan subtype A HW-l isolates (MVA/KEA- 1 through MVA/KEA-5), the 3 viruses expressing subtype A env alone (MVA/KEA-6 through MVA/KEA-8) and the MVA expressing subtype A gag/pol (MVA/KEA-9) were evaluated in an in-vivo mouse immunogenicity study
25 designed to measure the humoral and cellular immunogenicity of these vaccines. BALB/c mice (10 per group) were administered intraperitoneal immunizations of 107 infectious units of individual MVA/KEA viruses at weeks 0 and 3. Five mice per group were sacrificed two weeks after the 1st and 2nd immunizations and spleens were removed for assessment of cellular immunogenicity. Sera were collected from each mouse at weeks — 1,
30 0, 1, 2, 3, 4 and 5. Splenocytes and sera were pooled together by group. HW env-specific serum IgG responses were detected from all MVA/KEA-immunized groups after the 2nd immunization (Figure 38). While env-specific responses were detected in all groups except for the contiol group, they were strongest in MVA/KEA-3, 4, 5, 6 and 8. HW-specific cell-mediated immunity was assessed by three assays: (1) intracellular IFN- γ staining by flow cytometry (ICS), (2) IFN- γ secretion by ELISPOT, and (3) gag- peptide specific tetramer staining. (1) ICS: Splenocytes were collected two weeks after the 2nd immunization, stimulated for 7 days with MVA-infected P815 cells and then incubated overnight with P815 cells pulsed with a gag or pol peptide previously shown to be target of CD8 T cells in BALB/c mice (Casimiro et al. 2002 J Virol 76:185). Brefeldin A was included during the overnight incubation to prevent cytokine secretion. HW gag-specific responses were detected for each of the groups immunized with MVA/KEA viruses expressing env, gag and pol or gag and pol, but not control immunized, mice as evidenced by the production of intracellular IFN- γ after peptide stimulation (Figure 39). Splenocytes positive for HW gag ranged from 1% to 22% for the MVA/KEA-immunized mice. (2) IFN- γ ELISPOT: HW gag-specific IFN- γ responses from splenocytes without prior in- vitro stimulation were detected in all groups receiving MVA/KEA viruses expressing gag after the 1st immunization (Figure 40). (31 Tetramer staining: HW gag peptide-specific responses were measured by tetramer staining (H-2Kd gag LAI tetramer: AMQMLKETI, SEQ ID NO: 63) (Figure 41). Splenocytes were stained pre-WS. CD3 positive CD8 positive, tetramer positive cells were enumerated by flow cytometry. Similar to the above ICS and IFN-γ ELISPOT results, positive tetramer staining was observed for all of the groups immunized with MVA-KEA expressing gag. EXAMPLE 7
Construction of a Recombinant MVA Vaccine Expressing Subtype C HIV-1 Env,
Gag, and Pol for Use in Tanzania. As part ofthe overall program goal to conduct clinical vaccine trials in Africa, HIV- 1 sequences from Tanzania were selected. The predominant incident and prevalent HW-l subtype in Tanzania is subtype C. Several R5 subtype C HW-l strains were selected and used to prepare recombinant MVA vaccines expressing env, gag, protease and RT. One gagpol and one env clone from pure Tanzanian subtype C isolates were selected.
Steps described in EXAMPLE 5 were followed for the selected subtype C clones. These include: PCR amplification of truncated genes and cloning into pCR2.1.
Testing for in vitro expression. Testing for env and gag function.
Site directed mutagenesis in env to eliminate vaccinia virus early transcription termination sites.
Site directed mutagenesis in pol to inactivate enzymatic activity. Cloning into MVA shuttle plasmids pLAS-1 (gagpol) and pLAS-2 (env).
Recombination of gagpol into MVA 1974/NIH Clone 1 using primary CEF cells. Recombination of env into the recombinant virus expressing gag to produce a single virus expressing both gagpol and env.
Recombination of env into MVA 1974/NIH Clone 1 to produce a virus expressing env only.
The specifics of deletions and mutations in env and gagpol genes for TZC isolates is given in Table N. Plasmids and viruses expressing TZC env and gagpol genes are given in Table O and P.
Characterization of recombinant MVA/TZA viruses: The KEA viruses were characterized for gene expression. Immunoprecipitation of env and gag proteins is shown in Figure 42. BSC-1 cells were infected with individual recombinant viruses at a multiplicity of infection of 10, metabohcally labeled, and lysates were subjected to immunoprecipitation with a pool of sera from HW-l infected individuals. MVA/CMDR and MVA were included as positive and negative controls, respectively. The MVA/KEA virus expresses high levels of env and gag.
Virus-like particles were obtained by centrifugation of the supernatant of infected cells through a sucrose cushion (Karacostas et al. 1993 Virology 193:661). Gag p24 protein was found in the pelleted material indicating the formation of virus-like particles (Figure 43). EXAMPLE 8
Stability of expression of HIV-1 genes in recombinant MVA viruses. The stability of the inserted env and gagpol genes in recombinant viruses from each of the subtypes was tested after serial passage in CEF cells. Viruses were grown in CEF cells using a procedure that mimics that used for expansion of virus for large scale vaccine production, i.e. infection at low multiplicity, growth for 3 days at 37C, harvesting of virus from cell lysates. After repeated passage, the virus stocks were tested for stability of the inserts using a 3 -day immunostaining protocol. In this protocol, CEF cell monolayers were infected at a low multiplicity allowing for visualization of individual viral foci. After 3 days, the monolayers were fixed and then stained with monoclonal antibodies specific for either env or gag. Staining and non-staining foci were enumerated and results are shown in Table R, Very few non-staining foci were detected after 10-11 passages of each virus indicating that the inserted genes are stable after repeated passage in culture.
Table G. Oligonucleotides used for PCR amplification of env and gagpol genes
Table H(i) . Deletions/mutations in env genes
Table H(ii). Deletions/mutations in gagpol genes
Table I. UGD Plasmids
Table J. UGD Viruses
Table K(i). Deletions/mutations in env genes (KEA)
Table K(ii) Mutations/deletions in gagpol genes (KEA)
Table L. KEA Plasmids
Table M. KEA Viruses
Table N(i). Mutations/deletions in env genes (TZC)
Table O. TZC Plasmids
Table P. TZC Viruses
Table R. Stability of inserts in rMVA viruses
Appendix 1 - DNA sequences of gagpol and env genes from Ugandan HIV-1 clade D isolates:
99UGA03349 gagpol (SEQ ID NO: 51):
ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATTAGATGAATGGGAAAAAATTCGGTTA CGGCCAGGGGGAAACAAAAAATATAGATTAAAACATTTAGTATGGGCAAGCAGGGAGCTAGAA CGATTTGCACTTAATCCTGGTCTTTTAGAAACATCAGAAGGCTGTAGACAAATAATAGAACAGC TACAACCATCTATTCAGACAGGATCAGAGGAACTTAAATCATTACATAATACAGTAGTAACCCT CTATTGTGTACATGAAAGGATAAAGGTAGCAGATACCAAGGAAGCTTTAGATAAGATAAAGGA AGAACAAACCAAAAGTAAGAAAAAAGCACAGCAAGCAACAGCTGACAGCAGCCAGGTCAGCC AAAATTATCCTATAGTACAAAACCTACAGGGGCAAATGGTACACCAGTCCTTATCACCTAGGAC TTTGAATGCATGGGTAAAAGTAATAGAAGAGAAGGCTTTCAGCCCAGAAGTAATACCCATGTTT TCAGCATTATCAGAAGGAGCCACCCCAACAGATTTAAACACCATGCTAAACACAGTGGGGGGA CATCAAGCAGCCATGCAAATGTTAAAAGAGACTATCAATGAGGAAGCTGCAGAATGGGATAGG CTACATCCAGTGCCTGCAGGGCCTGTTGCACCAGGCCAAATGAGAGAACCAAGGGGAAGTGAT ATAGCAGGAACTACCAGTACCCTTCAGGAACAAATAGGATGGATGACAAGCAATCCACCTATCC CAGTAGGAGAAATCTATAAAAGATGGATAATCCTAGGATTAAATAAAATAGTAAGAATGTATA GCCCTGTCAGCATTTTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGATCG GTTCTATAAAACTCTACGAGCCGAGCAAGCTTCACAGGATGTAAAAAATTGGATGACTGAAACC TTGTTAGTCCAAAATGCGAATCCAGATTGTAAAACTATCTTAAAAGCATTGGGACCAGCGGCTA CATTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGACCCAGTCATAAAGCAAGAGTTT TGGCTGAGGCAATGAGCCAAGCATCAAACACAAATGCTGTTATAATGATGCAGAGGGGCAATTT CAAGGGCAAGAAAATCATTAAGTGTTTCAACTGTGGCAAAGAAGGACACCTAGCAAAAAATTG TAGGGCTCCTAGGAAAAGAGGCTGTTGGAAATGTGGAAAGGAAGGGCACCAAATGAAAGATTG TAATGAAAGACAGGCTAATTTTTTAGGGAGAATTTGGCCTTCCCACAAGGGGAGGCCAGGGAAT TTCCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGCAGAGAGCTTCGGGTTTGGGGAAGAG ATAACACCCTCCCAGAAACAGGAGGGGAAAGAGGAGCTGTATCCTTCAGCCTCCCTCAAATCAC TCTTTGGCAACGACCCCTAGTCACAATAAAAATAGGGGGACAGCTAAAGGAAGCTCTATTAGAT ACAGGAGCAGATGATACAGTAGTAGAAGAAATGAATTTGCCAGGAAAATGGAAACCAAAAATG ATAGGGGGAATTGGGGGCTTTATCAAAGTAAGACAGTATGATCAAATACTCGTAGAAATCTATG GATATAAGGCTACAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATTT GTTGACTCAGATTGGTTGCACTTTAAATTTTCCAATTAGTCCTATTGAAACTGTACCAGTAAAAT TAAAGTCAGGGATGGATGGTCCAAGAGTTAAACAATGGCCATTGACAGAAGAGAAAATAAAAG CACTAATAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAACTTTCAAGAATTGGACCTGAAA ATCCATACAATACTCCAATATTTGCCATAAAGAAAAAAGACAGTACTAAGTGGAGAAAATTAGT AGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAACTAGGAATACCACAT CCTGCAGGGCTAAAAAAGAAAAAATCAGTAACAGTACTGGAGGTGGGTGATGCATATTTTTCAG TTCCCTTATATGAAGACTTTAGAAAATACACTGCATTCACCATACCTAGTATAAACAATGAGAC ACCAGGAATTAGATATCAGTACAATGTGCTTCCACAAGGATGGAAAGGATCACCGGCAATATTC CAAAGTAGCATGACAAAAATTTTAGAACCTTTTAGAAAACAAAATCCAGAAGTGGTTATCTACC AATACATGCACGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAATAAAAATAGA GGAATTAAGGGGACACCTATTGAAGTGGGGATTTACCACACCAGACAAAAATCATCAGAAGGA ACCTCCATTTCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAAAAC TGCCAGAAAAAGAAAGCTGGACTGTCAATGATCTGCAGAAGTTAGTGGGGAAATTAAATTGGG CAAGTCAAATTTATTCAGGAATTAAAGTAAGACAATTATGCAAATGCCTTAGGGGAACCAAAGC ACTGACAGAAGTAGTACCACTGACAGAAGAAGCAGAATTAGAACTGGCAGAAAACAGGGAACT TCTAAAAGAAACAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAG AAACAAGGGCAAGACCAATGGACATATCAAATTTATCAAGAACAATATAAAAATTTGAAAACA GGAAAGTATGCAAAGAGGAGGAGTACCCACACTAATGATGTAAAACAATTAACAGAGGCAGTG CAAAAAATAGCCCAAGAATGTATAGTGATATGGGGAAAGACTCCTAAATTCAGACTACCCATAC AAAAGGAAACATGGGAAACATGGTGGACAGAGTATTGGCAGGCCACCTGGATTCCTGAGTGGG AGTTTGTCAATACCCCTCCCTTGGTTAAATTATGGTACCAGTTAGAGAAGGAACCCATAGTAGG AGCAGAAACCTTCTAA 99UGA07412 gagpol (SEQ ID NO: 52):
ATGGGTGCGAGAGCGTCAGTGTTAAGTGGGGGAAAATTAGATGAATGGGAAAGAATTCGGTTA CGGCCAGGGGGAAACAAAAGATATAAACTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAG CGATTTGCACTTAATCCTGGCCTTTTAGAAACATCAGAAGGCTGTAAACAAATATTGGGACAGC TACAACCAGCTATTCAGACAGGATCAGAAGAACTTAAATCATTATATAATACAGTAGCAACCCT CTATTGTGTACATGAGAGGCTAAAGGTAACAGACACCAAGGAAGCTTTAGACAAAATAGAGGA AGAACAAACCAAAAGTAAGAAAAAAGCACAGCAAGCAACAGCTGACACAAAAAACAGCAGCC AGGTCAGCCAAAATTATCCTATAGTACAAAACCTACAGGGGCAAATGGTACACCAGGCTATATC ACCTAGAACGTTGAACGCATGGGTAAAAGTAATAGAGGAGAAGGCTTTCAGCCCAGAAGTAAT ACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACA GTGGGGGGACATCAGGCAGCCATGCAGATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAA TGGGATAGGTTACATCCAGTACATGCAGGGCCTATTGCACCAGGACAAATGAGAGAACCAACA GGAAGTGATATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACCAGCAAT CCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTAGGATTAAATAAAATAGTAA GGATGTATAGCCCTGTCAGTATTTTGGACATAAAACAAGGGCCAAAGGAACCCTTTAGAGACTA TGTAGATCGGTTCTATAAAACTCTAAGGGCCGAGCAAGCTTCACAGGAGGTAAAAGGTTGGATG ACCGAAACCTTGTTGGTCCAAAATGCAAACCCAGATTGTAAAACCATCTTAAAAGCATTGGGAC CAGCGGCTACATTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGACCCGGTCATAAAG CAAGAGTTTTGGCTGAGGCAATGAGTCAAGTCTCAACAAATACTGCTATAATGATGCAGAGAGG CAATTTTAAGGGCCCAAAGAAAAGCATTAAGTGTTTTAACTGTGGCAAAGAAGGTCACACAGCA AAAAACTGTAGAGCTCCTAGGAAAAGGGGCTGTTGGAAATGTGGAAGGGAAGGACATCAAATG AAAGATTGCACTGAAAGACAGGCTAATTTTTTAGGGAAAATTTGGCCTTCCCACAAGGGAAGGC CAGGGAATTTCCTTCAGAACAGACCAGAGCCAACAGCCCCACCAGAAGAAAGCTTCGGGTTTGG GGAAGAGATAACACCCTCTCAGAAACAGGAGAAGAAGGACAAGGAGCTGTATCCTGTAGCTTC CCTCAAATCACTCTTTGGCAACGACCCCTTGTCACAATAAAGATAGGGGGACAGCTAAAGGAAG CTCTACTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATAAATTTGCCAGGAAAATGGA AACCAAAAATGATAGGGGGAATTGGAGGCTTTATCAAAGTAAGACAGTATGAGCAAATACTTG TAGAAATCTGTGGACAGAAAGCTATAGGTACAGTATTAGTAGGGCCTACACCTGTCAACATAAT TGGAAGAAATTTGTTGACTCAGATTGGTTGCACTTTAAATTTTCCAATTAGCCCTATTGAAACTG TACCAGTAAAATTAAAGCCAGGGATGGACGGTCCAAAAGTTAAACAATGGCCATTGACAGAAG AAAAGGTAAAAGCACTAATAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAGAA TTGGACCTGAAAATCCATACAATACTCCAATATTTGCCATAAAGAAAAAGGACAGTACTAAGTG GAGAAAATTAGTAGATTTCAGGGAACTTAATAAGAGAACTCAAGACTTCTGGGAAGTTCAACTA GGAATACCACATCCTGCGGGGCTAAAAAAGAAAAAATCAGTAACAGTACTGGAGGTGGGTGAT GCATATTTTTCAGTTCCCTTATATGAAGATTTTAGAAAATATACTGCATTCACCATACCTAGTAT AAACAATGAAACACCAGGAATTAGATATCAGTACAATGTGCTTCCACAAGGGTGGAAAGGATC ACCAGCAATATTCCAAAGTAGCATGACAAAAATCTTAGAACCTTTTAGAAAACAAAATCCAGAA ATGGTTATCTATCAATACATGCACGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATA GAATAAAAATAGAAGAATTAAGGGGACACCTGTTGAAGTGGGGATTTACCACACCAGACAAAA AGCATCAGAAAGAACCTCCATTTCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGT ACAGTCTATAAAACTGCCAGAACAAGAAAGCTGGACTGTCAATGATATACAGAAGTTAGTGGG AAAATTAAATTGGGCAAGCCAGATTTATCCAGGAATTAAGGTAAGACAATTATGCAAATGCATT AGGGGTACCAAAGCACTGACAGAAGTAGTACCACTGACAGAAGAAGCAGAATTAGAACTGGCA GAAAACAGGGAAATTCTAAGAGAACCAGTACATGGAGTGTATTATGACCCATCAAAAGACTTA ATAGCAGAGATACAGAAACAAGGGCAAGACCAGTGGACATACCAAATTTATCAAGAACAATAT AAAAATCTGAAAACAGGAAAGTATGCAAAAGTGAGGGGTACCCACACTAATGATGTAAAACAA TTAACAGAGGCAGTACAAAAAATAACCCAAGAATGTATAGTGATATGGGGAAAGCCTCCTAAA TTTAGACTACCCATACAAAAAGAAACATGGGAAATATGGTGGACAGAGTATTGGCAGGCCACCT GGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCTTTAGTTAAATTATGGTACCAATTAGAGAAG GAACCCATAGTAGGAGCAGAAACTTTCTAA 99UGA03349 envelope (SEQ ID NO: 53);
ATGAGAGTGAGGGGGATACAGAGGAACTATCAAAACTTGTGGAGATGGGGCACCTTGCTCCTTG GGATGTTGATGATATGTAAGQCTACAGAACAGTTGTGGGTCACAGTTTACTATGGGGTACCTGT GTGGAAAGAAGCAACCACTACTCTATTTTGTGCATCAGATGCTAAATCATATAAAGAAGAAGCA CATAATATCTGGGCTACACATGCCTGTGTACCAACAGACCCCAACCCACGAGAATTAATAATAG AAAATGTCACAGAAAACTTTAACATGTGGAAAAATAACATGGTGGAGCAGATGCATGAGGATA TAATCAGTTTATGGGATCAAAGCCTAAAACCATGTGTAAAATTAACCCCACTCTGTGTCACTTTA AACTGCACTGAATGGAGGAAGAATAACACTATCAATGCCACCAGAATAGAAATGAAAAACTGC TCTTTCAATCTAACCACAGAAATAAGAGATAGGAAAAAGCAAGTGCATGCACTTTTCTATAAAC TTGATGTGGTACCAATAGATGATAATAATAGTACTAATACCAGCTATAGGTTAATAAATTGTAA TACCTCAGCCATTACACAGGCGTGTCCAAAGGTAACCTTTGAGCCAATTCCCATACATTATTGTG CCCCAGCTGGATATGCGATTCTAAAATGTAACAATAAGAAGTTCAATGGGACAGGTCCATGCGA TAATGTCAGTACAGTACAGTGTACACATGGAATTAGGCCAGTAGTATCCACTCAATTGTTGTTGA ATGGCAGTCTAGCAGAAGAAGACATAATAATTAGATCTGAGAATCTCACAAATAATGCTAAAAT CATAATAGTACAGCTTAATGAGTCTGTAACAATTAATTGCACAAGGCCCTACAACAATACAAGA AGAGGTGTACATATAGGACCAGGGCGAGCATACTATACAACAGACATAATAGGAGATATAAGA CAAGCACATTGTAACATTAGTGGAGCAGAATGGAATAAGACTTTACATCGGGTAGCTAAAAAAT TAAGAGACCTATTTAAAAAGACAACAATAATTTTTAAACCGTCCTCCGGAGGGGACCCAGAAAT TACAACACACAGCTTTAATTGTAGAGGGGAATTCTTCTACTGCAATACAACAAGACTGTTTAAT AGCATATGGGGAAATAATAGTACAGGAGTTGATGAGAGTATAACACTCCCATGCAGAATAAAA CAAATTATAAACATGTGGCAGGGAGTAQGAAAAGCAATGTATGCCCCTCCCATTGAAGGACTAA TCAGCTGCTCATCAAATATTACAGGATTACTGTTGACAAGAGATGGTGGTGGAAGTAACAGTAG TCAGAATGAGACCTTCAGACCTGGAGGGGGAGATATGAGAGACAATTGGAGAAGTGAATTATA TAAATATAAAGTAGTAAGAATTGAACCATTAGGTCTAGCACCCTCCAAGGCAAAAAGAAGAGT AGTAGAAAGAGAGAAAAGAGCAATAGGACTAGGAGCTATGTTCCTTGGGTTCTTGGGAGCAGC AGGAAGCACGATGGGCGCAGCGTCACTGACGCTGACGGTACAGGCCAGACAGCTATTGTCTGGT ATAGTGCAACAGCAAAACAATTTGCTGAAGGCTATAGAGGCGCAACAGCACCTGTTGCAACTCA CAGTCTGGGGCGTTAAACAGCTCCAGGCAAGAGTCCTGGCTGTGGAAAGCTACCTAAGGGATCA ACAGCTCCTAGGAATTTGGGGTTGCTCTGGAAAACACATTTGCACCACCAATGTGCCCTGGAAC TCTAGCTGGAGTAATAAAACTCTAAAATCAATTTGGGATAACATGACCTGGATGGAGTGGGAAA GAGAAATTGACAATTACACAGGGATAATATACAATTTACTTGAAGAATCGCAAACCCAGCAAG AAAGAAATGAACAAGACCTATTGAAATTGGACCAATGGGCAAGTTTGTGGAATTGGTTTAGCAT AACAAAATGGCTGTGGTATATAAAAATATTTATAATGATAGTAGGAGGCTTGATAGGCTTAAGG ATAGTTTTTGCTGTGCTTTCTATAGTAAATAGAGTTAGGCAGGGATATTCACCTCTGTCGTTTCA GACCCTCCTCCCAGCCCCGCGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGA GCAAGGCTAA
99UGA07412 envelope (SEQ IS NO: 54):
ATGAGAGTGAGGGAGACAGTGAGGAATTATCAGCACTTGTGGAGATGGGGCATCATGCTCCTTG GGATGTTAATGATATGTAGTGCTGCAGACCAGCTGTGGGTCACAGTGTATTATGGGGTACCTGT GTGGAAAGAAGCAACCACTACTCTATTTTGTGCATCAGATGCTAAAGCACATAAAGCAGAGGCA CATAATATCTGGGCTACACATGCCTGTGTACCAACAGACCCCAATCCACGAGAAATAATACTAG GAAATGTCACAGAAAACTTTAACATGTGGAAGAATAACATGGTAGAGCAGATGCATGAGGATA TAATCAGTTTATGGGATCAAAGTCTAAAACCATGTGTAAAATTAACCCCACTCTGTGTTACTTTA AACTGCACTACATATTGGAATGGAACTTTACAGGGGAATGAAACTAAAGGGAAGAATAGAAGT GACATAATGACATGCTCTTTCAATATAACCACAGAAATAAGAGGTAGAAAGAAGCAAGAAACT GCACTTTTCTATAAACTTGATGTGGTACCACTAGAGGATAAGGATAGTAATAAGACTACCAACT ATAGCAGCTATAGATTAATAAATTGCAATACCTCAGTCGTGACACAGGCGTGTCCAAAAGTAAC CTTTGAGCCAATTCCCATACATTATTGTGCCCCAGCTGGATTTGCGATTCTGAAATGTAATAATA AGACGTTCAATGGAACGGGTCCATGCAAAAATGTCAGCACAGTACAGTGTACACATGGAATTAG GCCAGTAGTGTCAACTCAACTGTTGTTGAATGGCAGTCTAGCAGAAGAAGAGATAATAATTAGA TCTGAAAATATCACAAATAATGCAAAAACCATAATAGTACAGCTTAATGAGTCTGTAACAATTG ATTGCATAAGGCCCAACAACAATACAAGAAAAAGTATACGCATAGGACCAGGGCAAGCACTCT ATACAACAGACATAATAGGGAATATAAGACAAGCACATTGTAATGTTAGTAAAGTAAAATGGG GAAGAATGTTAAAAAGGGTAGCTGAAAAATTAAAAGACCTTCTTAACCAGACAAAGAACATAA CTTTTGAACCATCCTCAGGAGGGGACCCAGAAATTACAACACACAGCTTTAATTGTGGAGGGGA ATTCTTCTACTGCAATACATCAGGACTATTTAATGGGAGTCTGCTTAATGAGCAGTTTAATGAGA CATCAAATGATACTCTCACACTCCAATGCAGAATAAAACAAATTATAAACATGTGGCAAGGAGT AGGAAAAGCAATGTATGCCCCTCCCATTGCAGGACCAATCAGCTGTTCATCAAATATTACAGGA CTATTGTTGACAAGAGATGGTGGTAATACTGGTAATGATTCAGAGATCTTCAGACCTGGAGGGG GAGATATGAGAGACAATTGGAGAAGTGAATTATACAAATATAAAGTAGTAAGAATTGAACCAA TGGGTCTAGCACCCACCAGGGCAAAAAGAAGAGTGGTGGAAAGAGAAAAAAGAGCAATAGGA CTGGGAGCTATGTTCCTTGGGTTCTTGGGAGCGGCAGGAAGCACGATGGGCGCAGCGTCACTGA CGCTGACGGTACAGGCCAGACAGTTATTGTCTGGTATAGTGCAACAGCAAAACAATTTGCTGAG AGCTATAGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATTAAACAGCTCCAGGCA AGAGTCCTGGCTATGGAAAGCTACCTAAAGGATCAACAGCTCCTAGGAATTTGGGGTTGCTCTG GAAAACACATTTGCACCACTACTGTGCCCTGGAACTCTACCTGGAGTAATAGATCTGTAGAGGA GATTTGGAATAATATGACCTGGATGCAGTGGGAAAGAGAAATTGAGAATTACACAGGTTTAATA TACACCTTAATTGAAGAATCGCAAACCCAGCAAGAAAAGAATGAACAAGAACTATTGCAATTG GATAAATGGGCAAGTTTGTGGAATTGGTTTAGTATAACAAAATGGCTGTGGTATATAAAAATAT TCATAATGATAGTAGGAGGCTTAATAGGTTTAAGAATAGTTTTTGCTGTGCTTTCTTTAGTAAAT AGAGTTAGGCAGGGATATTCACCTCTGTCTTTTCAGACCCTCCTCCCAGCCCCGAGGGGACCCG ACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGCAAGGCTAA
98UG57128 envelope (SEQ ID NO: 55):
ATGAGAGTGAGGGGGATAGAGAGGAATTATCAGCACTTATGGTGGAGATGGGGCACCATGCTC CTTGGGATATTGATGATATGTAGTGCTGCAGAACAATTGTGGGTCACAGTTTATTATGGGGTACC TGTGTGGAAAGAAGCAACCACTACTCTATTTTGTGCATCAGATGCTAAAGCATATAAAGCAGAG GCACACAATATCTGGGCTACACATGCCTGTGTACCAACAGACCCCAACCCACAAGAAATAGTAC TAGAAAATGTCACAGAAAACTTTAACATGTGGAAAAATAGCATGGTGGAGCAGATGCATGAGG ATGTAATCAGTTTATGGGATCAAAGCCTAAAACCATGTGTAAAATTAACCCCACTCTGTGTCACT TTAAACTGCACTAATGCCACTGCCACTAATGCCACTGCCACTAGTCAAAATAGCACTGATGGTA GTAATAAAACTGTT CACAGACACAGGAATGAAAAACTGCTCTTTCAATGTAACCACAGATCT AAAAGATAAGAAGAGGCAAGACTATGCACTTTTCTATAAACTTGATGTGGTACGAATAGATGAT AAGAATACCAATGGTACTAATACCAACTATAGATTAATAAATTGTAATACCTCAGCCATTACAC AAGCGTGTCCAAAGATAACCTTTGAGCCAATTCCCATACATTATTGTGCCCCAGCTGGATATGCG ATTCTAAAATGTAATAATAAGACATTCAATGGGACGGGTCCATGCAAAAACGTCAGCACAGTAC AGTGTACACATGGGATTAGGCCAGTAGTGTCAACTCAACTGTTGTTGAATGGCAGTCTAGCAGA GGAAGAGATAGTAATTAGATCTGAAAACCTCACAAATAATGCTAAAATTATAATAGTACAGCTT AATGAAGCTGTAACAATTAATTGCACAAGACCCTCCAACAATACAAGACGAAGTGTACATATAG GACCAGGGCAAGCAATCTATTCAACAGGACAAATAATAGGAGATATAAGAAAAGCACATTGTA ATATTAGTAGAAAAGAATGGAATAGCACCTTACAACAGGTAACTAAAAAATTAGGAAGCCTGTT TAACACAACAAAAATAATTTTTAATGCATCCTCGGGAGGGGACCCAGAAATTACAACACACAGC TTTAATTGTAACGGGGAATTCTTCTACTGCAATACAGCAGGACTGTTTAATAGTACATGGAACA GGACAAATAGTGAATGGATAAATAGTAAATGGACAAATAAGACAGAAGATGTAAATATCACAC TTCAATGCAGAATAAAACAAATTATAAACATGTGGCAGGGAGTAGGAAAAGCAATGTATGCCC CTCCCGTTAGTGGAATAATCCGATGTTCATCAAATATTACAGGACTGTTGCTGACAAGAGATGG TGGTGGTGCAGATAATAATAGGCAGAATGAGACCTTCAGACCTGGGGGAGGAGATATGAGAGA CAATTGGAGAAGTGAATTATACAAATATAAAGTAGTAAGAATTGAACCACTAGGTATAGCACCC ACCAAGGCAAGGAGAAGAGTGGTGGAAAGAGAAAAAAGAGCAATAGGACTGGGAGCCTTGTT CCTTGGGTTCTTGGGAACAGCAGGAAGCACGATGGGCGCAGTGTCAATGACGCTGACGGTACAG GCCAGACAAGTATTGTCTGGTATAGTGCAACAGCAAAACAATCTGCTGAGGGCTATAGAGGCGC AACAGCATCTGTTGCAACTCACAGTCTGGGGCATTAAACAGCTCCAGGCAAGAATCCTGGCTGT GGAAAGCTACCTAAAGGATCAACAGCTCCTAGGAATTTGGGGTTGCTCTGGAAAACACATTTGC ACCACTAATGTGCCCTGGAACTCTAGCTGGAGTAATAAATCTCTAAATTATATTTGGAATAACAT GACCTGGATGGAGTGGGAAAAGGAAATTGACAATTACACAGAATTAATATACAGCTTAATTGA AGTATCGCAAATCCAGCAAGAAAAGAATGAACAAGAACTATTGAAATTGGACAGTTGGGCAAG TTTGTGGAATTGGTTTAGCATAACAAAATGGCTGTGGTATATAAAAATATTCATAATGATAGTA GGAGGCTTGATAGGCTTAAGAATAGTTTTTGCTGTGCTTTCTTTAGTAAATAGAGTTAGGCAGGG ATACTCACCTCTGTCGTTTCAGACCCTTATCCCAGCCTCGAGGGGΛCCCGACAGGCCCGAAGGA ACAGAAGGAGAAGGTGGAGAGCAAGGCTAA
Appendix 2 - DNA sequences of MVA shuttle plasmids: pLAS-1 (SEQ ID NO: 56): GAATTCGTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAGTAAAACATG GCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAACCATGTTGTGAAAAAGTCAAGAA CGTTCACATTGGCGGACAATCTAAAAACAATACAGTGATTGCAGATTTGCCATATATGGATAAT GCGGTATCCGATGTATGCAATTCACTGTATAAAAAGAATGTATCAAGAATATCCAGATTTGCTA ATTTGATAAAGATAGATGACGATGACAAGACTCCTACTGGTGTATATAATTATTTTAAACCTAA AGATGCCATTCCTGTTATTATATCCATAGGAAAGGATAGAGATGTTTGTGAACTATTAATCTCAT CTGATAAAGCGTGTGCGTGTATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGGATGTT TCCTTTTTTACCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATTTCTCTATCGATGCGGCA CCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTATATCTAGACACCAGCGTCTACATGACGA GCTTCCGAGTTCCAATTGGTTCAAGTTTTACATAAGTATAAAGTCCGACTATTGTTCTATATTAT ATATGGTTGTTGATGGATCTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAATATTAG CAAAAATATATTAGACAATACTACAATTAACGATGAGTGTAGATGCTGTTATTTTGAACCACAG ATTAGGATTCTTGATAGAGATGAGATGCTCAATGGATCATCGTGTGATATGAACAGACATTGTA TTATGATGAATTTACCTGATGTAGGCGAATTTGGATCTAGTATGTTGGGGAAATATGAACCTGAC ATGATTAAGATTGCTCTTTCGGTGGCTGGGTACCAGGCGCGCCTTTCATTTTGTTTTTTTCTATGC TATAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGG CAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTG ACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCT GAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAA CAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGAT CCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAG ACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCT CGGCATGCACGAGCTGTACAAGTAAGAGCTCGGTTGTTGATGGATCTGTGATGCATGCAATAGC TGATAATAGAACTTACGCAAATATTAGCAAAAATATATTAGACAATACTACAATTAACGATGAG TGTAGATGCTGTTATTTTGAACCACAGATTAGGATTCTTGATAGAGATGAGATGCTCAATGGATC ATCGTGTGATATGAACAGACATTGTATTATGATGAATTTACCTGATGTAGGCGAATTTGGATCTA GTATGTTGGGGAAATATGAACCTGACATGATTAAGΛTTGCTCTTTCGGTGGCTGGCGGCCCGCTC GAGGCCGCTGGTACCCAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAA ATTGAAAGCGAGAAATAATCATAAATAAGCCCGGGGATCCTCTAGAGTCGACCTGCAGGGAAA GTTTTATAGGTAGTTGATAGAACAAAATACATAATTTTGTAAAAATAAATCACTTTTTATACTAA TATGACACGATTACCAATACTTTTGTTACTAATATCATTAGTATACGCTACACCTTTTCCTCAGAC ATCTAAAAAAATAGGTGATGATGCAACTTTATCATGTAATCGAAATAATACAAATGACTACGTT GTTATGAGTGCTTGGTATAAGGAGCCCAATTCCATTATTCTTTTAGCTGCTAAAAGCGACGTCTT GTATTTTGATAATTATACCAAGGATAAAATATCTTACGACTCTCCATACGATGATCTAGTTACAA CTATCACAATTAAATCATTGACTGCTAGAGATGCCGGTACTTATGTATGTGCATTCTTTATGACA TCGCCTACAAATGACACTGATAAAGTAGATTATGAAGAATACTCCACAGAGTTGATTGTAAATA CAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTACACATTCACCAGAAACTAGTTA AGCTTGTCTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGT GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC TGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCGAGTC GGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT ATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGC GGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAA GAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT TTTCGATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGT TCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTT GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAA AGGATCTCAAGAAGATCCTTTGATCTTT CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA ATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTC GTGTAGATAACTAeGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAqCGCGAG ACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGGCATTGCTACAGGCATCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCC CCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAG ATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA GTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCA CGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCG GAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCA GCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAG TGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACG CCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAG TCACGACGTTGTAAAACGACGGCCAGTGAATTGGATTTAGGTGACACTATA
pLAS -2 (SEQ ID NO: 57):
CCTCCTGAAAAACTGGAATTTAATACACCATTTGTGTTCATCATCAGACATGATATTACTGGATT TATATTGTTTATGGGTAAGGTAGAATCTCCTTAATATGGGTACGGTGTAAGGAATCATTATTTTA TTTATATTGATGGGTACGTGAAATCTGAATTTTCTTAATAAATATTATTTTTATTAAATGTGTATA TGTTGTTTTGCGATAGCCATGTATCTACTAATCAGATCTATTAGAGATATTATTAATTCTGGTGC AATATGACAAAAATTATACACTAATTAGCGTCTCGTTTCAGACATGGATCTGTCACGAATTAATA CTTGGAAGTCTAAGCAGCTGAAAAGCTTTCTCTCTAGCAAAGATGCATTTAAGGCGGATGTCCA TGGACATAGTGCCTTGTATTATGCAATAGCTGATAATAACGTGCGTCTAGTATGTACGTTGTTGA ACGCTGGAGCATTGAAAAATCTTCTAGAGAATGAATTTCCATTACATCAGGCAGCCACATTGGA AGATACCAAAATAGTAAAGATTTTGCTATTCAGTGGACTGGATGATTCGAGGTACCAGGCGCGC CCTTTCATTTTGTTTTTTTCTATGCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT GGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGC CCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCC GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCA CCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACAC CCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCAC TACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCA CCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGT GACCGCCGCCGGGATCACTCTCGGCATGCACGAGCTGTACAAGTAAGAGCTCGCTTTCTCTCTA GCAAAGATGCATTTAAGGCGGATGTCCATGGACATAGTGCCTTGTATTATGCAATAGCTGATAA TAACGTGCGTCTAGTATGTACGTTGTTGAACGCTGGAGCATTGAAAAATCTTCTAGAGAATGAA TTTCCATTACATCAGGCAGCCACATTGGAAGATACCAAAATAGTAAAGATTTTGCTATTCAGTG GACTGGATGATTCTCCGGATGGTACCCAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAG GGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATAAGCCCGGGGATCCTCTAGAGTCGACC TGCAGGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGGCTTGGGGGGCTGC AGGTGGATGCGATCATGΛCGTCCTCTGCAATGGATAACAATGAACCTAAAGTACTAGAAATGGT ATATGATGCTACAATTTTACCCGAAGGTAGTAGCATGGATTGTATAAACAGACACATCAATATG TGTATACAACGCACCTATAGTTCTAGTATAATTGCCATATTGGATAGATTCCTAATGATGAACAA GGATGAACTAAATAATACACAGTGTCATATAATTAAAGAATTTATGACATACGAACAAATGGCG ATTGACCATTATGGAGAATATGTAAACGCTATTCTATATCAAATTCGTAAAAGACCTAATCAAC ATCACACCATTAATCTGTTTAAAAAAATAAAAAGAACCCGGTATGACACTTTTAAAGTGGATCC CGTAGAATTCGTAAAAAAAGTTATCGGATTTGTATCTATCTTGAACAAATATAAACCGGTTTATA GTTACGTCCTGTACGAGAACGTCCTGTACGATGAGTTCAAATGTTTCATTGACTACGTGGAAACT AAGTATTTCTAAAATTAATGATGCATTAATTTTTGTATTGATTCTCAATCCTAAAAACTAAAATA TGAATAAGTATTAAACATAGCGGTGTACTAATTGATTTAACATAAAAAATAGTTGTTAACTAAT CATGAGGACTCTACTTATTAGATATATTCTTTGGAGAAATGACAACGATCAAACCGGGCATGCA AGCTTGTCTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGT GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC TGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCGAGTC GGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT ATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGC GGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAA GAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT TTTCGATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGT TCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTT GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA AACAAACCACCGCTGGTAGCGGTGGTITTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAA AGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA ATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTC GTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAG ACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGGCATTGCTACAGGCATCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCC CCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAG ATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA GTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCA CGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCG GAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCA GCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAG TGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACG CCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAG TCACGACGTTGTAAAACGACGGCCAGTGAATTGGATTTAGGTGACACTATAGAATACGAATTC
Appendix 3- DNA sequences of gagpol and env genes from Kenyan HW-l clade A isolates:
KER2008 gagpol (SEQ ID NO: 58):
ATGGGTGCGAGAGCGTCAGTATTAAGTGGGGGAAAATTAGATGCATGGGAGAAAATTCGGTTA AGGCCAGGGGGAAAGAAAAAATATAGACTGAAACACTTAGTATGGGCAAGCAGGGAGCTGGA AAAATTCGTACTTAACCCTAGCCTTTTAGAAACTTCAGAAGGATGTCAGCAAATAATGAACCAA ATACAACCAGCTCTTCAGACAGGAACAGAAGAACTTAGATCATTATTTAATGCAGTAGCAACCC TCTATTGTGTACATCAACGGATAGAGGTAAAAGACACCAAGGAAGCTTTAGATAAAGTAGAGG AAATACAAAACAAGAGCAAGCAAAAGACACAACAGGCAGCAGCTGATACAGGAAACAACAGC AAGGTCAGCCATAATTACCCTATAGTGCAAAATGCACAAGGGCAAATGATACATCAGTCCTTAT CACCAAGGACTTTGAATGCATGGGTAAAGGTAATAGAAGAAAGGGGTTTCAGCCCAGAAGTAA TACCCATGTTCTCAGCATTATCAGAAGGAGCCATCCCACAAGATTTAAATATGATGCTGAACAT AGTGGGGGGACACCAGGCAGCTATGCAAATGTTAAAAGAAACTATCAATGAGGAAGCTGCAGA ATGGGACAGGTTACATCCAGCACAGGCAGGGCCTATTCCACCAGGCCAGATAAGAGACCCAAG GGGAAGTGACATAGCAGGAACTACTAGTACCCCTCAGGAACAAATAACATGGATGACAAACAA CCCACCTATCCCAGTGGGAGACATCTATAAAAGATGGATAATCCTAGGATTAAATAAAATAGTA AGAATGTATAGCCCTGTTAGCATTTTAGATATAAAACAGGGGCCAAAAGAACCCTTCAGAGACT ATGTAGATAGGTTCTTTAAAGTTCTCAGAGCCGAACAAGCTACACAGGAAGTAAAAGGCTGGAT GACAGAGACCCTGCTGGTTCAAAATGCAAATCCAGATTGTAAGTCCATTTTAAGAGCATTAGGA ACAGGGGCTACATTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGAGGACCCGGCCATAAA GCAAGGGTTTTAGCTGAGGCAATGAGTCAAGCACAACAGGCAAATGTAATGATGCAGAGGGGC AGCTTTAAGGGGCAGAAAAGAATTAAGTGCTTCAACTGTGGCAAAGAGGGACACCTAGCCAGA AATTGCAGAGCCCCTAGGAAAAAAGGCTGTTGGAAGTGTGGGAAAGAAGGACACCAAATGAAA GATTGCAATGAGAGACAGGCTAATTTTTTAGGGAAAATTTGGCCTTCCAGCAAGGGGAGGCCAG GAAATTTTCCCCAGAGCAGACCGGAGCCAACAGCCCCACCAGCAGAGATCTTTGGGATGGGGG AAGAGATAACCTCCCCTCCGAAGCAGGAGCAGAAAGAGAGGGAACAAACCCCACCCTTTGTTT CCCTCAAATCACTCTTTGGCAACGACCCGTTGTCACAGTAAAAGTAGGAGGAGAAATGAGAGAA GCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGATATAAATTTGCCAGGAAAATGGA AACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAGGTAAAACAATATGATCAGGTATCTAT AGAAATTTGTGGAAAAAAGGCTATAGGTACGGTATTAGTAGGACCTACACCTGTCAACATAATT GGAAGAAATATGTTGACTCAGATTGGTTGTACCTTAAATTTTCCAATTAGTCCTATTGAGACTGT ACCAGTAACATTAAAGCCAGGAATGGATGGCCCAAGGGTTAAACAATGGCCATTGACAGAAGA GAAAATAAAΛGCATTGACAGAAATTTGTAAAGAGATGGAAAAGGAAGGAAAAATTTCAAAAAT TGGGCCTGAAAATCCATACAATACTCCAATATTTGCAATAAAGAAAAAAGATAGCACTAAATGG AGGAAATTAGTAGATTTCAGAGAGCTCAATAAAAGAACACAAGACTTTTGGGAAGTTCAATTAG GGATACCGCATCCAGCGGGCCTAAAAAAGAAAAAATCAGTAACAGTACTAGAGGTGGGGGATG CATATTTTTCAGTTCCCCTAGATAAAAACTTTAGAAAGTATACTGCATTTACCATACCTAGTTTA AATAATGAAACACCAGGAATCAGGTATCAGTACAATGTGCTTCCACAAGGATGGAAAGGATCA CCAGCAATATTCCAGTGCAGTATGACAAAAATCTTAGAGCCCTTTAGATCAAAAAATCCAGAAA TAATTATCTATCAATACATGCACGACTTGTATGTAGGATCAGATTTAGAAATAGGGCAGCATAG AGCAAAAATAGAAGAATTAAGAGCTCATCTACTGAGCTGGGGATTTACTACACCAGACAAAAA GCATCAGAAAGAACCTCCATTCCTTTGGATGGGATATGAGCTCCATCCTGACAAGTGGACAGTC CAGCCTATAGAGCTGCCAGAAAAAGAAAGCTGGACTGTCAATGATATACAGAAATTAGTGGGA AAACTAAATTGGGCCAGTCAAATTTATCCAGGAATTAAAGTAAAGCAATTGTGTAAACTTCTCA GGGGAGCCAAAGCCCTAACAGATATAGTAACACTGACTGAGGAAGCAGAATTAGAATTAGCAG AGAACAGGGAGATTCTAAAAGACCCTGTGCATGGGGTATATTATGACCCATCAAAAGACTTAAT AGCAGAAATACAGAAACAAGGGCAAGACCAATGGACATACCAAATTTATCAGGAGCCATTTAA AAATCTAAAAACAGGAAAATATGCAAGAAAAAGGTCTGCTCACACTAATGATGTAAGACAATT AGCAGAAGTAGTGCAGAAAGTGGTCATGGAAAGCATAGTAATATGGGGAAAGACTCCTAAATT TAAACTACCCATACAAAAAGAGACATGGGAGACATGGTGGATGGACTATTGGCAAGCTACCTG GATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCCTAGTAAAATTATGGTACCAGTTAGAGAAA GACCCCATAGCAGGAGCAGAGACTTTCTAA KNH1144 envelope (SEQ ID NO: 59):
ATGAGAGTGATGGGGATACAGATGAATTGTCAGCACTTATTGAGATGGGGAACTATGATCTTGG GATTGATAATAATCTGTAATGCTGTAAACAGCAACTTGTGGGTTACTGTCTATTATGGGGTACCT GTGTGGAAAGATGCAGAGACCACCTTATTTTGTGCATCAGATGCTAAAGCATATAAAACAGAAA AGCATAATGTCTGGGCTACACATGCCTGTGTGCCCACAGACCCCAACCCACAAGAAATACCTTT GGAAAATGTGACAGAAGAGTTTAACATGTGGAAAAATAAAATGGTAGAACAAATGCATACAGA TATAATCAGTCTATGGGACCAAAGCCTACAGCCATGTGTAAAGTTAACCCCTCTCTGCATTACTT TAAACTGTACAGATGTTACTAATGTTACAGATGTTAGTGGTACGAGGGGCAACATCACCATCAT GAAAGAGATGGAGGGAGAAATAAAAAACTGTTCTTTCAATATGACCACAGAAATAAGGGATAA GAAACAGAAAGTATATTCACTCTTTTATAGACTTGATGTAGTACCAATAAATCAGGGTAATAGT AGTAGTAAAAACAGTAGTGAGTATAGATTAATAAGTTGTAATACCTCAGCCATTACACAAGCTT GCCCAAAGGTAAGCTTTGAGCCAATTCCCATACATTATTGTGCCCCAGCTGGTTTTGCGATCCTG AAGTGTAGGGATAAGGAGTTCAATGGAACAGGGGAATGCAAGAATGTCAGCACAGTCCAATGC ACACATGGAATCAAGCCAGTAGTATCAACTCAACTACTGTTAAATGGCAGTCTAGCAGAAGAAA AGGTAAAAATCAGAACTGAAAATATCACAAACAATGCCAAAACTATAGTAGTACAACTTGTCG AGCCTGTGAGAATTAATTGTACTAGACCTAATAACAATACAAGAGAGAGTGTGCGTATAGGGCC AGGACAAGCATTCTTTGCAACAGGTGACATAATAGGGGATATAAGACAAGCACATTGTAATGTC AGTAGATCACAATGGAATAAGACTTTACAACAGGTAGCTGAACAATTAAGAGAACACTTTAAA AACAAAACAATAATATTTAACAGTTCCTCAGGAGGGGATCTAGAAATCACAACACATAGTTTCA ATTGTGGAGGAGAATTCTTCTATTGTAATACATCAGGTCTGTTCAATAGCACCTGGAATACCAGC ATGTCAGGGTCAAGTAACACGGAGACAAATGACACTATAACTCTCCAATGCAGAATAAAGCAA ATTATAAATATGTGGCAGAGAACAGGACAAGCAATATATGCCCCTCCCATCCAGGGAGTGATAA GGTGTGAATCAAACATCACAGGACTACTGTTAACAAGAGATGGTGGGGAGGAGAAGAACAGTA CAAATGAAATCTTCAGACCTGGAGGAGGAGATATGAGGGACAACTGGAGAAGTGAATTATATA AGTATAAAGTAGTAAAAATTGAACCACTAGGAGTAGCACCCACCAGGGCAAGGAGAAGAGTGG TGGGAAGAGAAAAAAGAGCAGTTGGAATAGGAGCTGTTTTCCTTGGGTTCTTAGGAGCAGCAG GAAGCACTATGGGCGCGGCGTCAATAACGCTGACGGTACAGGCCAGGCAATTATTGTCTGGCAT AGTGCAGCAGCAGAGCAATTTGCTGAGGGCTATAGAGGCTCAACAACATATGTTGAAACTCACG GTCTGGGGCATTAAACAGCTCCAGGCAAGAGTCCTTGCTGTGGAAAGATACCTAAGGGATCAAC AGCTCCTAGGAATTTGGQGCTGCTCTGGAAAACTCATCTGCACCACTAATGTGCCCTGGAACTCT AGTTGGAGTAATAAATCTCAGGATGAAATATGGAACAACATGACCTGGCTGCAATGGGATAAA GAAATTAGCAATTACATAAACCTAATATATAGTCTAATTGAAGAATCGCAAAACCAGCAGGAAA AGAATGAACAAGACTTATTGGCATTGGGCAAGTGGGCAAATCTGTGGACTTGGTTTGACATATC AAATTGGCTGTGGTATATAAGΛΛTATTTATAATGATAGTAGGAGGCTTAATAGGATTAAGAATA GTTTTTGCTGTGCTTGCTGTAATΛAAGAGAGTTAGGCAGGGATACTCACCTGTGTCATTTCAGAT CCATGCCCCAAACCCAGGGGGTCTCGACAGGCCCGGAAGAATCGAAGGAGAAGGTGGAGAGCA AGACTAA
KNH1207 envelope (SEQ ID NO: 60)
ATGAGAGTGATGGGGATACAGATGAATTGTCAAAGCTTGTGGAGATGGGGAACTATGATCTTGG GAATGTTAATGATTTGTAGTGTTGCAGGAAACTTGTGGGTTACTGTCTACTATGGGGTACCTGTG TGGAAAGAGGCAGACACCACCTTATTTTGTGTATCAAATGCTAGAGCATATGATACAGAAGTGC ATAATGTCTGGGCTACACATGCCTGTGTACCTACGGACCCCAACCCACAAGAAATAGATTTGGA GAATGTGACAGAAGAGTTTAACATGTGGAAAAATAACATGGTAGAGCAAATGCATACAGATAT AATTAGTCTATGGGACCAAAGCCTAAAACCATGTGTAAAGTTAACCCCTCTCTGCGTTACTTTAG ATTGTGGCTATAATGTAACCAACTTGAATTTCAGCAGTAACATGAAAGGAGACATAACAAACTG CTCTTACAATATGACCACAGAAATAAGGGATAGGAAACAGAAAGTGTATTCACTTTTCTATAGG CTTGATATAGTACCAATTAATGAAGAAAAGAATAATAGCAGGGAGACTAGTCCGTATAGATTAA TAAATTGTAATACCTCAGCCATTACACAAGCTTGTCCTAAGGTATCTTTTGAACCAATTCCCATA CATTATTGTGCCCCAGCCGGTTTTGCGATTCTAAAATGTAAGGATGCAGAGTTCAATGGAACAG GGCCATGCAAGAATGTCAGCACAGTACAATGTACACATGGAATCAGGCCAGTAATATCAACTCA ACTGCTGTTAAATGGCAGTTTAGCAGAGAATGGGACAAAGATTAGATCTGAAAATATCACAAAC AATGCCAAAACCATAATAGTACAACTTAACGAGACCGTACAAATTAATTGTACCAGACCTAGCA ACAATACAAGAAAAAGTGTACGTATAGGACCAGGACAAGCATTCTATACAACAGGTGATATAA CAGGGGATATAAGACAAGCATATTGTAATGTCAGTAGACAAGAATGGGAACAAGCATTAAAAG GGGTAGTTATACAATTAAGAAAACACTTTAACAAAACAATAATCTTTAACAGTTCCTCAGGAGG GGATTTAGAAATTACAACACATAGTTTTAATTGTGGAGGAGAATTCTTCTATTGTGATACATCAG GCCTGTTTAATAGCACCTGGAACACGAACACCACCGAGCCAAACAACACAACGTCAAATGGCA CTATCATTCTCCAATGCAGAATAAAGCAAATTATAAATCTGTGGCAGAGAACCGGACAAGCAAT GTATGCCCCTCCCATCCAAGGGGTAATAAGGTGTGATTCCAACATTACAGGACTACTATTAACA AGAGATGGTGGAGTAGTTGATAGTATAAATGAAACCGAAATCTTCAGACCTGGAGGAGGAGAT ATGAGGGACAATTGGAGAAGTGAATTATATAAGTATAAAGTAGTAAAAATTGAACCACTAGGA GTAGCACCCACCGGGGCAAAGAGAAGAGTGGTGGAGAGAGAAAAAAGAGCAGTTGGCATAGG AGCTGTATTCATTGGGTTCTTAGGAGCAGCAGGAAGCACTATGGGCGCGGCGTCAATAACGCTG ACGGTACAGGCCAGACAATTATTGTCTGGCATAGTGCAACAGCAAAGCAATTTGCTGAGGGCTA TAGAGGCTCAACAGCATATGTTGAGACTCACGGTCTGGGGCATTAAGCAGCTCCAGGCAAGAGT CCTGGCTGTGGAAAGATACCTAAGGGATCAACAGCTCCTAGGAATTTGGGGCTGCTCTGGAAAA CTCATCTGCACCACTAATGTGCCCTGGAACTCTAGTTGGAGTAATAAATCTCAGGAGGAAATAT GGGGTAACATGACCTGGCTGCAATGGGATAAAGAAATTAGCAATTACACACAAACAATATATA ACCTACTTGAAGAATCGCAGAACCAGCAGGAAAAGAATGAACAAGACTTATTGGCATTGGACA AGTGGGCAAATTTGCGGACTTGGTTTGACATAACAAATTGGCTGTGGTATATAAAAATGTTTAT AATGATAGTAGGAGGCTTAATAGGATTAAGAATAGTTTTTGCTGTGCTTTCTGTAATAAATAGA GTTAGGCAGGGATACTCACCTCTGTCGTTTCAGACCCATATCCCGAGCCCAAGGGGTCTCGATA GGCCCGGAAGAATCGAAGGAGAAGGTGGAGAGCAAGACTAA
Appendix 4 - DNA sequences of gagpol and env genes from Tanzanian HIV-1 clade C isolates:
TZA-246 gagpol (SEQ ID NO: 61):
ATGGGTGCGAGAGCGTCAATATTAAGAGGGGGAAAATTAGATCGATGGGAAAAAATTAGGTTA AGGCCAGGGGGAAAGAAAAGCTATATGATAAAACACTTAGTATGGGCAAGCAGGGAGCTGGAA AGATTTGCACTTAACCCTAGCCTTTTAGAGACATCAGAAGGCTGTAAACAAATAATGAAACAGC TACAACCAGCTCTTCAGACAGGAACAGAAGAACTTAAATCATTATTCAATGCAATAGCAGTTCT CTATTGTGTACATGAAGGGATAGATGTAAAAGACACCAAGGAAGCCTTAGACAAGATAGAGGA AGAACAGAACAAAAGTCAGCAAAAAACACAGCAGGCAGAAGCAGCTGGCGGAAAAGTCAGTC AAAATTATCCTATAGTGCAGAATCTCCAAGGACAAATGGTACACCAGTCCATATCACCTAGAAC TTTGAATGCATGGGTAAAAGTAATAGAGGAAAAGGCTTTTAGCCCAGAGGTAATACCCATGTTT ACAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAATACAGTGGGGGGA CATCAAGCAGCCATGCAAATGTTAAAAGATACCATCAATGAGGAGGCTGCAGAATGGGATAGG ATACATCCAGTACATGCAGGGCCTACTGCACCAGGCCAAATGAGAGAACCAAGGGGAAGTGAC ATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGCATGGATGACAGCTAACCCACCTGTTC CAGTGGGAGAAATCTACAAAAGATGGATAATACTGGGTTTAAATAAAATAGTAAGAATGTATA GCCCTGTCAGCATTTTGGACATAAAACAAGGGCCAAAGGAACCCTTTAGAGACTATGTAGATCG GTTCTTTAAAACTTTAAGAGCTGAACAGGCTACACAAGATGTAAAAAATTGGATGACAGACACC TTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACCATTTTAAGAGCATTAGGACCAGGGGCTA CATTAGAAGAAATGATGACAGCATGTCAAGGAGTGGGAGGACCTGGCCACAAAGCCAGAGTTT TGGCTGAGGCAATGAGCCAAGCAAACACACACATAATGATGCAGAGAAGCAATTTTAAAGGCT CTAAAAGAATTGTTAAATGTTTCAACTGTGGCAAGGAAGGGCACATAGCCAGAAATTGCAGGGC CCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGACTGTACTGA GAGGCAGGCTAATTTTTTAGGGAAAATTTGGCCTTCCCACAAGGGGAGGCCAGGGAATTTCCTT CAGAACAGGTCAGAGCCAACAGCCCCACCAACGAACAGGCCAGAGCCAACAGCTCCACCAGCA GAGAGCTTCAGGTTCGAGGAAGCAACCCCTGCTCCGAAGCAGGAGCTGAAAGACAGGGAACCT TTAATTTCCCTCAAATCACTCTTTGGCAGCGACCCCTCGTCTCAATAAAAGTAGGGGGTCAAACA AAGGAGGCTCTTTTAGACACAGGAGCAGATGATACAGTATTAGAAGAAATAAATTTGCCAGGA AAATGGAAACCCAAAATGATAGGAGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAG ATAGTTATAGAAATTTGTGGAAAAAAGGCTATAGGTACAGTATTAGTAGGACCCACCCCTGTCA ACATAATTGGAAGAAATATGTTGACTCAGCTTGGATGCACACTAAATTTTCCAATTAGTCCTATT GAAACTGTACCAGTAAAGTTAAAGCCAGGAATGGATGGCCCAAAGGTTAAACAATGGCCATTG ACAGAAGAAAAAATAAAGGCATTAACAGCAATTTGTGAAGAAATGGAGAAGGAAGGAAAAAT TACAAAGATTGGGCCTGAAAATCCATATAACACTCCAGTATTTGCCATAAAAAAGAAGGACAGT ACTAAGTGGAGAAAATTAGTAGATTTCAGGGAACGCAATAAAAGAACTCAAGATTTTTGGGAA GTTCAATTAGGCATACCACACCCAGCAGGGTTAAAAAAGAAAAAATCAGTGACAGTACTGGAG GTGGGGGATGCATACTTCTCAGTTCCTTTAGATGAAGGCTTCAGGAAATATACTGCATTCACCAT ACCTAGTATAAACAATGAAACACCAGGAATTAGATATCAATACAATGTGCTTCCACAGGGATGG AAAGGATCACCAGCAATATTCCAGAGTAGCATGACAAAAATCTTAGAGCCCTTTAGAGCACAAA ATCCAGAAATAGTCATCTATCAATATATGCACGACTTATATGTAGGATCTGACTTAGAAATAGG GCAACATAGAGCAAAAATAGAGGAATTAAGAGAACATCTATTAAAGTGGGGATTTACCACACC AGACAAGAAACATCAGAAAGAACCCCCATTTCTTTGGATGGGGTATGAACTCCATCCTGACAAA TGGACAGTACAGCCTATAACGCTGCCAGAAAAGGAAAGCTGGACTGTCAATGATATACAGAAG TTAGTGGGAAAACTAAACTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAACTGTATA AACTCCTTAGGGGAGCCAAAGCACTAACAGACATAGTACCACTAACTGAAGAGGCAGAATTAG AATTGGCAGAGAACAGGGAAATTCTAAAAGAACCAGTACATGGGGTATATTATGACCCATCAA AAGACTTGATAGCTGAAATACAGAAACAAGGGCATGACCAATGGACATATCAAATTTACCAAG AACCATTCAAAAATCTGAAAACAGGGAAGTATGCAAAAATGAGGAGTGCCCACACTAATGATG TAAAACAATTAACAGAGGCAGTGCAAAAAATAGCCATGGAAGGCATAGTAATATGGGGAAAGA CTCCTAAATTTAGACTGCCCATTCAAAAGGAAACATGGGAAACATGGTGGACAGACTATTGGCA AGCCACCTGGATTCCTGAGTGGGAGTTTGTTAATACCCCTCCCCTAGTAAAATTATGGTACCAGC TGGAGAAAGAACCCATAGTAGGAGCAGAAACTTTC TZA-125 envelope (SEQ ID NO: 62):
ATGAGAGTGAAGGGGATATTGAGGAATTGGCAACACAGGTGGATATGGATCTGGATCATCTTAG GCTTTTGGATGCTAATGATTTQTAATGGGAACTTGTGGGTCACTGTCTACTATGGGGTACCTGTG TGGAAAGAAGCAAATGCTCCTCTATTTTGTGCATCAGATGCTAAAGCATATGAGAAAGAAGTGC ATAATGTCTGGGCTACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAACTAGACTTGGT AAATGTAACAGAAAATTTTAACATGTGGAAAAATGACATGGTAGATCAGATGCATGAGGATAT AATCAGTTTATGGGATGAAAGCCTAAAGCCATGTGTAAAGTTGACCCCACTCTGTGTCACTCTA AACTGTACTAATGCTAATATTAATAATGATACTGTTGCTAATAGTGGTACTTTTAAGGTTGATAA TAGTAGTAATGTAGTAAAAAATTGCTCTTTCAATATAACCACAGAAATAAGAGATAAGAAGAAA AAAGAATATTCATTGTTTTATAGACTTGATATATTACCACTTGATAACTCTAGTGAGTCTAAGAA CTATAGTGAGTATGTATTAATAAATTGTAATGCCTCAACCGTAACACAAGCCTGTCCAAAGGTCT CTTTTGACCCAATTCCTATACATTATTGTGCTCCAGCTGGTTATGCGATTCTAAAGTGTAAAGAT AAGACATTCAATGGAACAGGACCATGCAGTAATGTCAGCACAGTACTATGTACACATGGAATTA AGCCAGTGGTATCAACTCAATTACTGTTAAATGGTAGCCTAGCAGAAGAAGGGATAGTAATTAG ATCTGAAAATCTGACAAACAATGCCAAAACAACAATAGTACAGCTTAATGAACCTGTAGAAATT ATGTGTGTAAGACCCGGCAATAATACAAGAAAAAGTGTGAGGATAGGACCAGGACAAACATTC TATGCAACAGGAGGCATAATAGGAGATATAAGACAAGCACATTGTAACATTAGTAGAAGTGAT TGGAATAAAACTTTACAAGAGGTAGGTAAAAAATTACGAGAATACTTCCACAATAAAACAATA AGATTTAAACCGGCGGTCGTAGGAGGGGACCTGGAAATTACAACACATAGCTTTAATTGTAGAG GAGAATTCTTCTATTGCAATACATCAGAACTGTTTACAGGTGAATATAATGGTACTGAGTATAA GAATACTTCAAATTCAAATCCTAACATCACACTCCCATGTAGAATAAAACAATTTGTAAACATG TGGCAGAGGGTAGGACGAGCAATGTATGCCCCTCCTATTGAAGGAAACATAACATGTAACTCAA GTATCACAGGACTACTATTGACATGGGATGGAGGAAACAATACTAATGGCACAGAGACATTTAG ACCTGGAGGAGGAGATATGAGGGATAATTGGAGAAGTGAATTATATAAATATAAAGTGGTAGA AΛTTAAACCATTAGGAATAGCACCCACTAGTGCAAAAAGGAGAGTGGTGGAGAGAGAGAAAAG AGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTAGGAGCAGCAGGAAGCACTATGGGCGCA GCATCAATAACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAACAGCAAAGCA ATTTGCTGAGGGCCATAGAGGCGCAACAGCATATGTTGCAACTCACAGTCTGGGGCATTAAACA GCTCCAGACAAGAGTCCTGGCTATAGAAAGATACCTAAAGGATCAACAGCTCCTAGGGATTTGG GGCTGCTCTGGAAAACTCATCTGCACCACTGCTGTGCCTTGGAACACTAGTTGGAGTAATAAAA CTGAACAGGACATTTGGAATCTAACCTGGATGCAGTGGGATAGAGAAGTTAGTAATTACACAGA CATAATATACAGGTTGCTTGAAGACTCACAAATCCAGCAGGAAAACAATGAAAAGGATTTACTA GCATTGGACAGTTGGAAAAATCTGTGGAATTGGTTTGACATAACAAATTGGTTGTGGTATATAA GAACATTCATAATGATAGTAGGAGGCTTGATAGGCTTAAGGATAATTTTTGCTGTAATTTCTATA GTGAATAGAGTTAGGCAGGGATACTCACCTTTGTCATTTCAGACCCTTACCCCAACCCCGAGGG GACCAGAAAGGCTCGGAGGAATCGAAGAAGAAGGTGGAGAGCAAGACTAA
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While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope ofthe invention. All figures, tables, and appendices, as well as patents, applications, and publications, referenced to above, are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS:
1. A pharmaceutical composition comprising a recombinant MVA virus expressing an HW env, gag, and pol gene or modified gene thereof for production of an HW Env, Gag, and Pol antigen by expression from said recombinant MVA virus, wherein said HW env gene is modified to encode an HW Env protein composed of gpl 20 and the membrane-spanning and ectodomain of gp41 but lacking part or all of the cytoplasmic domain of gp41, and a pharmaceutically acceptable carrier, wherein said HW env, gag, and pol genes are isolatable from an individual infected with Ugandan clade D isolate 99UGA03349, 99UGA07412, or 98UG57128.
2. The pharmaceutical composition of claim 1 comprising 99UGA03349 gagpol in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
3. The pharmaceutical composition of claim 1 comprising 99UGA07412 gagpol in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
4. The pharmaceutical composition of claim 1 comprising 99UGA03349 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
5. The pharmaceutical composition of claim 1 comprising 99UGA07412 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
6. The pharmaceutical composition of claim 1 comprising 98UG57128 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
7. The pharmaceutical composition of claim 1, wherein said recombinant
MVA virus is MVA/UGD- 1 defined as comprising 99UGA07412 gagpol in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto, and 99UGA07412 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
8. The pharmaceutical composition of claim 1, wherein said recombinant
MVA virus is MVA/UGD-2 defined as comprising 99UGA03349 gagpol in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto, and 98UG57128 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
9. The pharmaceutical composition of claim 1, wherein said recombinant MVA virus is MVA/UGD-3 defined as comprising 99UGA07412 gagpol in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto, and 99UGA03349 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
10. The pharmaceutical composition of claim 1, wherein said recombinant MVA virus is MVA/UGD-4 defined as comprising 99UGA03349 gagpol in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto, and 99UGA07412 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
11. The pharmaceutical composition of claim 1, wherein said recombinant MVA virus is MVA/UGD-5 defined as comprising 99UGA03349 gagpol in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto, and 98UG57128 envelope in Appendix 1 or sequence having at least about 90%, 95% or 99.9% identity thereto.
12. The pharmaceutical composition of claim 1 wherein said recombinant MVA virus additionally expresses an additional HW gene or modified gene thereof for production of an HW antigen by expression from said recombinant MVA virus, wherein said additional HW gene is a member selected from the group consisting of vif, vpr, tat, rev, vpu, and nef.
13. An MVA shuttle plasmid comprising pLAS-1 of Appendix 2 or sequence having at least about 90%, 95% or 99.9% identity thereto, or pLAS-2 of Appendix 2 or sequence having at least about 90%, 95% or 99.9% identity thereto.
14. A method of making a recombinant MVA virus comprising preparing the MVA shuttle plasmid of Claim 13 and combining said MVA shuttle plasmid with a MVA virus to produce said recombinant MVA virus, and isolating said recombinant MVA virus.
15. A method of boosting a CD8+ T cell immune response to an HW Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a composition of claim 1, whereby a CD8+ T cell immune response to the antigen previously primed in the primate is boosted.
16. A method of inducing a CD8+ T cell immune response to an HW Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a composition of claim 1, whereby a CD8+ T cell immune response to the antigen in the primate is induced.
17. A method of inducing a CD8+ T cell immune response to an HW Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a priming composition comprising nucleic acid encoding said antigen and then provision in the primate of a boosting composition which comprises claim 1, whereby a CD8+ T cell immune response to the antigen is induced.
18. The method of claim 15 , wherein the primate is a human.
19. The method of claim 15, wherein administration of the recombinant MVA virus is by needleless injection.
20. The method of claim 15, wherein the priming composition comprises plasmid DNA encoding said antigen.
21. MVA 1974/NIH Clone 1.
22. A pharmaceutical composition comprising a recombinant MVA virus expressing an HW env, gag, and pol gene or modified gene thereof for production of an HW Env, Gag, and Pol antigen by expression fiOin said recombinant MVA virus, wherein said HW env gene is modified to encode an HW Env protein composed of gpl 20 and the membrane-spanning and ectodomain of gp41 but lacking part or all of the cytoplasmic domain of gp41, and a pharmaceutically acceptable carrier, wherein said HW env, gag, and pol genes are isolatable from an individual infected with Kenyan clade A isolate OOKE- KER2008, 00KE-KNH1144, or 00KE-KNH1207.
23. The pharmaceutical composition of claim 22 comprising 00KE-KER2008 gagpol in Appendix 3 or sequence having at least about 90%, 95 %> or 99.9% identity thereto.
24. The pharmaceutical composition of claim 22 comprising 00KE-KNH1144 envelope in Appendix 3 or sequence having at least about 90%, 95%> or 99.9%> identity thereto.
25. The pharmaceutical composition of claim 22 comprising 00KE-KNH1207 envelope in Appendix 3 or sequence having at least about 90%>, 95%> or 99.9%> identity thereto.
26. The pharmaceutical composition of claim 22, wherein said recombinant MVA virus is MVA/KEA- 1 defined as comprising 00KE-KER2008 gagpol in Appendix 3 or sequence having at least about 90%, 95% or 99.9%> identity thereto, and 00KE- KNH1144 envelope in Appendix 3 or sequence having at least about 90%ι, 95% or 99.9%) identity thereto.
27. The pharmaceutical composition of claim 22, wherein said recombinant MVA virus is MVA/KEA-2 defined as comprising 00KE-KER2008 gagpol in Appendix 3 or sequence having at least about 90%, 95%> or 99.9%> identity thereto, and 00KE- KNH1207 envelope in Appendix 3 or sequence having at least about 90%, 95% or 99.9%) identity thereto .
28. The pharmaceutical composition of claim 22, wherein said recombinant MVA virus is MVA/KEA-3 defined as comprising 00KE-KER2008 gagpol in Appendix 3 or sequence having at least about 90%, 95%> or 99.9% identity thereto, and 00KE- KNH1144 envelope in Appendix 3 or sequence having at least about 90%, 95%> or 99.9% identity thereto.
29. The pharmaceutical composition of claim 22, wherein said recombinant MVA virus is MVA/KEA-4 defined as comprising 00KE-KER2008 gagpol in Appendix 3or sequence having at least about 90%>, 95% or 99.9% identity thereto, and 00KE- KNH1144 envelope in Appendix 3 or sequence having at least about 90%, 95%> or 99.9% identity thereto.
30. The pharmaceutical composition of claim 22, wherein said recombinant MVA virus is MVA KEA-5 defined as comprising 00KE-KER2008 gagpol in Appendix 3 or sequence having at least about 90%, 95% or 99.9%o identity thereto, and 00KE- KNH1144 envelope in Appendix 3 or sequence having at least about 90%, 95%> or 99.9% identity thereto.
31. The pharmaceutical composition of claim 22 wherein said recombinant MVA virus additionally expresses an additional HW gene or modified gene thereof for production of an HW antigen by expression from said recombinant MVA virus, wherein said additional HW gene is a member selected from the group consisting of vif, vpr, tat, rev, vpu, and nef
32. A method of boosting a CD8+ T cell immune response to an HW Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a composition of claim 22, whereby a CD8+ T cell immune response to the antigen previously primed in the primate i2s boosted.
33. A method of inducing a CD8+ T cell immune response to an HIV Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a composition of claim 22, whereby a CD8+ T cell immune response to the antigen in the primate is induced.
34. A method of inducing a CD8+ T cell immune response to an HIV Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a priming composition comprising nucleic acid encoding said antigen and then provision in the primate of a boosting composition which comprises claim 22, whereby a CD8+ T cell immune response to the antigen is induced.
35. The method of claim 32, wherein the primate is a human.
36. The method of claim 32, wherein administration of the recombinant MVA virus is by needleless injection.
37. The method of claim 32, wherein the priming composition comprises plasmid DNA encoding said antigen.
38. A pharmaceutical composition comprising a recombinant MVA virus expressing an HIV env, gag, and pol gene or modified gene thereof for production of an HW Env, Gag, and Pol antigen by expression from said recombinant MVA virus, wherein said HW env gene is modified to encode an HW Env protein composed of gpl 20 and the membrane-spanning and ectodomain of gp41 but lacking part or all of the cytoplasmic domain of gp41, and a pharmaceutically acceptable canier, wherein said HW env, gag, and pol genes are isolatable from an individual infected with Tanzanian clade C isolate 00TZA- 246 or 00TZA-125.
39. The pharmaceutical composition of claim 38 comprising 00TZA-246 gagpol in Appendix 4 or sequence having at least about 90%, 95% or 99.9%o identity thereto.
40. The pharmaceutical composition of claim 38 comprising 00TZA-125 envelope in Appendix 4 or sequence having at least about 90%., 95% or 99.9% identity thereto.
41. The pharmaceutical composition of claim 38, wherein said recombinant
MVA virus is MVA/TZC-1 defined as comprising 00TZA-246 gagpol in Appendix 4 or sequence having at least about 90%, 95% or 99.9% identity thereto, and 00TZA-125 envelope in Appendix 4 or sequence having at least about 90%, 95% or 99.9% identity thereto.
42. The pharmaceutical composition of claim 38 wherein said recombinant MVA virus additionally expresses an additional HIV gene or modified gene thereof for production of an HW antigen by expression from said recombinant MVA virus, wherein said additional HIV gene is a member selected from the group consisting of vif, vpr, tat, rev, vpu, and nef.
43. A method of boosting a CD8+ T cell immune response to an HW Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a composition of claim 38, whereby a CD8+ T cell immune response to the antigen previously primed in the primate is boosted.
44. A method of inducing a CD8+ T cell immune response to an HW Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a composition of claim 38, whereby a CD8+ T cell immune response to the antigen in the primate is induced.
45. A method of inducing a CD8+ T cell immune response to an HW Env, Gag, or Pol antigen in a primate, the method comprising provision in the primate of a priming composition comprising nucleic acid encoding said antigen and then provision in the primate of a boosting composition which comprises claim 38, whereby a CD8 T cell immune response to the antigen is induced.
46. The method of claim 43, wherein the primate is a human.
47. The method of claim 43, wherein administration of the recombinant MVA virus is by needleless injection.
48. The method of claim 43, wherein the priming composition comprises plasmid DNA encoding said antigen.
EP04758588A 2003-03-28 2004-03-29 Mva virus expressing modified hiv envelope, gag, and pol genes Expired - Lifetime EP1610816B1 (en)

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WO2004087201A3 (en) 2005-03-31
EP2363143A3 (en) 2012-02-15
AU2004226345A1 (en) 2004-10-14
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CA2520637A1 (en) 2004-10-14
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US7303754B2 (en) 2007-12-04
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